Micro MR device including cell culture micro chamber and method for manufacturing the same

Abstract

An MR device (100) comprising a miniature magnetic resonance system (101) and a cell culture chamber (502) for analyzing biological samples with a size less than about 1000 μm, wherein the device (100) is at least partially covered by a passivating bonding layer (800). The invention also relates to a method for manufacturing the device (100), comprising a step of depositing a thin passivating bonding layer (800) on the system (101). The deposition step is preferably performed by a deposition process selected from chemical vapor deposition and physical vapor deposition.

Description

Technical Field

[0001] The invention belongs to the field of nuclear magnetic resonance spectroscopy for analyzing microscopic objects. Background Art

[0002] Nuclear Magnetic Resonance (NMR) and Electron Spin Resonance (ESR) (often generically denoted as Magnetic Resonance (MR)) are widely known spectroscopic tools for chemical analysis of intact bulk materials. The use of these spectroscopic tools requires a static external magnetic field B0. In such an environment, the electrons and nuclei of the sample can respond to excitation in a collective manner through a magnetic field oscillating at the so-called Larmor frequency v0 = (γ / 2π) B0, where γ is the gyromagnetic ratio of the target nucleus or electron. The effective value of the gyromagnetic ratio γeff depends on the type of nucleus (including isotopes) and the chemical environment in which the nucleus and / or electron is embedded. In fact, for a given chemical environment, the shielding of the external magnetic field is different, resulting in slightly different Larmor frequencies. This phenomenon of Larmor frequency separation due to the chemical configuration of the target nucleus and / or electron embedded therein is called chemical shift. NMR and ESR experiments, which can be performed in either pulsed or continuous excitation schemes, allow for the quantitative examination of the different chemical shifts present within a sample, resulting in the ability to obtain chemical information at potentially very high resolution.

[0003] A relevant characteristic of NMR (and to some extent ESR) technology is biocompatibility. Such unique characteristics are due to the use of relatively low frequencies to produce resonance phenomena as needed. Due to the relatively low frequencies in play, the excitation of NMR can be carried out on intact living organisms, such as those with weak interference with biomolecules and processes, thereby avoiding any biological changes in the sample. Today, NMR is routinely used to examine living animals and humans for research and medical diagnostic purposes. The value of the gyromagnetic ratio of protons is about 42.5775 MHz / T. The use of NMR for in vivo medical diagnosis is currently approved for humans, with field values ​​up to 7T (corresponding to a 1H Larmor frequency of about 300 MHz).

[0004] The MR instruments currently available on the market have been optimized for examining solid, biological or liquid samples down to a volume as small as 1 μL. Such instruments employ inductors made with metal conductors as excitation and sensing elements. Such inductors come in many shapes and forms, each suitable for a particular sample and / or application (e.g., solenoid-type coils, Helmholtz coils, and saddle coils).

[0005] In order to apply MR technology to the analysis of microscopic samples, microfabrication of inductors has been previously proposed and implemented. Some examples of such prior art can be found in references [1 to 3] and are described in US 2011 / 0091987. In this method, a micro-coil (i.e., a microcoil) is connected to a passive component (usually a capacitor) to achieve a structure that resonates at the Larmor frequency. Such a resonator is then connected to standard transmit-receive (TX / RX) electronics via a cable. In this method, the leads connecting the microcoil to external components and electronics form parasitic inductors, which greatly complicates the application of MR to small samples. In addition to introducing losses that reduce the effective signal-to-noise ratio and detect signals from areas other than the area occupied by the sample, such parasitic structures also introduce unwanted signals when the experimenter introduces components or structures intended to limit the sample. In view of these limitations, microcoils have not yet provided a universal solution for the MR field at a scale below 1 μL.

[0006] Another approach that has been recently proposed is what is referred to herein as an "ultra-compact" MR device, in which the necessary TX (transmit) and / or RX (receive) electronics are placed very close to the excitation and / or sensing elements (e.g., microcoils). An example of an ultra-compact MR device is a complementary metal-oxide-semiconductor (CMOS) microchip in which the TX / RX electronics and the microcoil are co-integrated on the same substrate. Some examples of such devices are described in [4 to 7]. Ultra-compact MR devices offer the advantages of having almost no parasitic sensitive volumes and being able to easily implement state-of-the-art MR at volume scales of 5 pL up to 1 μL.

[0007] A currently unmet challenge is to create a universal sample handling strategy that allows the user to easily manipulate and contain samples, particularly solid samples, such as embryos (e.g., mammals, aquatic species, plants), eukaryotic cells or bacteria, 3D cell cultures, stem cell organoids, microtissues of any kind, in very small volumes of liquid media (i.e., less than about 1 μL), in close proximity to the sensing element (i.e., as close as 1 nm), typically 5 to 1000 μm in size, and at the same time provide a controlled and biocompatible environment for the biological entity being analyzed. Assuming the use of an ultra-compact MR device, a comprehensive structure is needed that is designed to provide the following: biocompatible and controllable conditions, universal sample handling strategies, and robustness against intensive use and exposure to selected liquids (e.g., culture media for cell growth and / or development). Summary of the invention

[0008] The present invention solves and overcomes the above disadvantages by providing an ultra-compact (i.e., having a localized sensitive volume near the active element of the sensor) magnetic resonance probe contained in a housing and combined with a microstructure to realize a magnetic resonance biological culture chamber that can be suitable for living microorganisms and cells. The use of the present invention may involve biochemical studies and / or clinical diagnosis involving biological samples such as embryos (e.g., mammals, aquatic species, plants), eukaryotic cells or bacteria, 3D cell cultures, stem cell organoids, any kind of microtissue. The use of the present invention may also involve magnetic sensors.

[0009] One of the key challenges faced and addressed by the present inventors was to create a system comprising a miniaturized magnetic resonance device effectively coupled to a biological culture microchamber, optimized for analyzing biological samples less than about 1000 μm in size, i.e., providing excellent spin sensitivity for the sample under study (e.g., 2.10 μm in a 1 nL volume). 13 Spin / Hz 1 / 2 To this end, fabrication methods have been proposed that appropriately exploit deposition processes to provide conformal layers that 1) protect the ultracompact sensing device (passivation), 2) secure the microstructure to the substrate (binding), and 3) potentially provide a biocompatible ground and variant (e.g., a functionalized surface for cell immobilization and / or capture).

[0010] Thus, in a first aspect, there is provided herein an MR device for analyzing biological samples having a size less than about 1000 μm comprising a miniature magnetic resonance system and a cell culture chamber, the device comprising a passivating bonding layer at least partially covering the system. The invention also relates to a method for manufacturing the device, the method comprising the step of depositing a thin passivating bonding layer on the system. In a preferred embodiment, the deposition is performed by a deposition process selected from chemical vapor deposition and / or physical vapor deposition.

[0011] The cell culture chamber preferably has a volume of 0.1 nL to 1 μL and is integrated into the system.

[0012] Advantageously, the cell culture microchamber comprises a floor consisting of said layer.

[0013] The above and other objects, features and advantages of the present invention will become more apparent through a study of the following description with reference to the accompanying drawings showing some preferred aspects of the subject matter. However, the present invention is not limited to the embodiments described below and / or shown in the accompanying drawings; on the contrary, the scope of the present invention is defined by the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A schematic diagram of one embodiment of a complete magnetic resonance apparatus is shown, in which an NMR microsystem (referred to as μMRBM) is inserted into a socket and into a magnet for use;

[0016] Figure 2 a shows a schematic top view of two embodiments of ultra-compact sensor geometries, including a gasket, active components, sensing and / or excitation elements. Figure 2 b shows a 3D rendering of such an ultra-compact sensor mounted on a printed circuit board.

[0017] Figure 3 a and Figure 3 b shows a schematic cross-sectional view (side view) of some embodiments of a magnetic resonance biological culture microchamber mounted on a printed circuit board. Figure 3 In a, the μMRBM has no cover for the housing and the hosting microstructure, whereas Figure 3 b, with such a cover. Figure 3 c shows a 3D rendering of an ultra-compact sensor combined with a host microstructure mounted on a printed circuit board.

[0018] Figure 4 3D rendering showing a basic example of an "external auxiliary coil" to supplement the ultra-compact sensor. Figure 4 The gradient coil is depicted in a. Figure 4 b The planar coil is placed in close proximity to the sensing element. Such a coil can be used to implement both pulse gradients or excitation pulses.

[0019] Figure 5 Two basic examples of internal auxiliary coils are illustrated. Figure 5 In a, a portion of the coil is fabricated using wire bonding and metal on chip. Figure 5 b shows the case of an auxiliary coil that is strongly coupled to the sensing element and is fully realized with available metal layers on the microchip hosting the ultra-compact sensor.

[0020] Figure 6 Two examples of arrayed ultracompact sensor and host microstructures that can be used as a basis for fabricating arrayed μMRBMs are shown. Figure 6 a shows an array in which individual ultra-compact sensors are placed on the same printed circuit board, each sensor being connected to a microstructure. Figure 6 b shows an array where a single microchip substrate contains multiple ultracompact sensors and a single body microstructure individually targets each excitation and / or detection area. DETAILED DESCRIPTION

[0021] The subject matter described herein will be illustrated below by the following description of those aspects depicted in the accompanying drawings. However, it should be understood that the subject matter described in this specification is not limited to the aspects described below and depicted in the accompanying drawings; rather, the scope of the subject matter described herein is defined by the claims. Furthermore, it should be understood that the specific conditions or parameters described and / or shown below do not limit the subject matter described herein, and that the terminology used herein is used only for the purpose of describing specific aspects by way of example and is not intended to be limiting.

[0022] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. In addition, nouns not modified with quantifiers include one or more, unless the context otherwise requires. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in a plurality of general and more specific references cited and discussed throughout this specification. In addition, for clarity, the use of the term "about" is intended to cover variations of + / -10% of a given value herein.

[0023] The following description will be better understood through the following definitions.

[0024] Unless the context clearly indicates otherwise, nouns used below and in the appended claims that are not modified by quantifiers include one / kind or more / kind. In addition, unless otherwise specified, the use of "or / or" means "and / or". Similarly, "comprising" and "including" are interchangeable and are not intended to be limiting. It should also be understood that where the term "comprising / including" is used to describe multiple embodiments, those skilled in the art will understand that in some specific cases, the language "essentially consisting of..." or "consisting of..." can be used to describe the embodiments alternatively.

[0025] "Thin film or layer" means in this article a solid element whose thickness is much smaller than other dimensions (e.g., at least one-fifth compared to other dimensions). It is a solid layer with an upper surface and a bottom surface, with any suitable shape and nanometer-level or even micrometer-level thickness as required and circumstance (e.g., for producing its manufacturing step). In some preferred embodiments, the thickness of the film according to the present invention is about 1nm to 1000 microns, preferably about 1nm to 600 microns, about 1nm to 300 microns, about 1nm to 100 microns, about 1nm to 50 microns, about 100nm to 100 microns, about 1 micron to 500 microns, about 1 micron to 300 microns, about 1 micron to 100 microns, about 10 microns to 100 microns, or about 1 micron to 50 microns.

[0026] The term "passivation" refers to the behavior of a film or layer according to the present invention, which acts as an isolating shielding material, preventing physical, chemical and / or electrical connection between two or more elements of a device (e.g. an electronic component and a liquid medium), thereby avoiding, for example, short circuits and corrosion caused by, for example, oxidation of metal components.

[0027] "Deposition" means the action of applying a thin film to a surface in this article, which foresees any technique of depositing a thin film of material onto a substrate or a previously deposited layer. "Thin" is a relative term, but most deposition techniques control the layer thickness to within tens of nanometers. The deposition method may include one of chemical vapor deposition, physical vapor deposition, spin coating, and spray coating. In one embodiment according to the present invention, the deposition method includes a chemical vapor deposition process. In an alternative or additional embodiment, the deposition method includes a physical vapor deposition process.

[0028] The "chemical vapor deposition" process or "CVD" is a deposition method that is usually used to produce high-quality, high-performance solid materials under vacuum. In a typical CVD, a substrate or wafer is exposed to one or more volatile precursors, which react and / or decompose on the substrate surface to produce the desired deposit. CVD is commonly used to deposit conformal films and increase substrate surfaces in ways that more traditional surface modification techniques cannot achieve. When depositing very thin layers of material, CVD is extremely useful in so-called atomic layer deposition. In the framework of the present invention, chemical vapor deposition is intended to include atmospheric pressure CVD (APCVD), low-pressure CVD (LPCVD) and ultrahigh vacuum CVD (UHVCVD). Furthermore, in the framework of the present invention, chemical vapor deposition is intended to include plasma treatment methods such as microwave plasma-assisted CVD (MPCVD), plasma-enhanced CVD (PECVD), remote plasma-enhanced CVD (RPECVD), atomic-layer CVD (ALCVD), combustion chemical vapor deposition (CCVD), hot filament CVD (HFCVD), hybrid physical-chemical vapor deposition (HPCVD), rapid thermal CVD (RTCVD), vapor-phase epitaxy (VPE), photo-initiated CVD (PICVD) and laser chemical vapor deposition (LCVD).

[0029] The "physical vapor deposition" process or "PVD" generally describes a variety of vacuum deposition methods that can be used to produce thin films and coatings. PVD is characterized by a process in which a material changes from a condensed phase to a gas phase and then back to a thin film condensed phase, thereby depositing a layer of material on a solid substrate atom by atom or molecule by molecule. In the framework of the present invention, physical vapor deposition is intended to include cathodic arc deposition, electron beam physical vapor deposition, evaporative deposition or evaporation, sputtering deposition, sublimation sandwich method, ion beam assisted deposition and hybrid physical chemical vapor deposition (HPCVD).

[0030] "Active elements" in this context means a set of means for exciting and / or detecting resonance, which may consist of electronic devices and / or optical elements (eg lasers, on-chip light sources, plasmonic structures or microlenses).

[0031] By "sensing element" is meant herein an element of the system of the invention which serves as a signal pick-up prior to the necessary amplification operations (and ultimately frequency down-conversion). In some cases, such as when the element consists of an inductor, the sensing element may be used for both NMR excitation and detection, either for excitation purposes only or for detection purposes only. For this reason, the sensing element is also referred to herein as a "sensing and / or excitation element". The "sensing element" of the ultra-compact device defines the area in which sensing is performed, its geometrical properties, the limits of performance in terms of sensing power, the method used to perform the sensing.

[0032] To date, there are two physical phenomena that can be exploited to realize such a sensing element, namely induction and optomagnetic phenomena. In the inductive sensing method, the sensor consists of an inductor realized with a metal winding. In this implementation, the sample magnetization dynamics, which is a typical feature of magnetic resonance, is converted into a voltage signal by Faraday's law. In operation, the sample must be placed in a region of space where the inductor is able to generate a magnetic field when some current flows through the inductor above the space. The current flowing through the coil generates a magnetic flux, which can generate a voltage if it is intercepted by a second adjacent coil. In such a case, the two coils are said to be coupled. The "coupling coefficient k" is a dimensionless number between 0 and 1, which represents the fraction of the magnetic flux generated by one coil and intercepted by the second coil. This number depends strongly on the relative distance and orientation between the two coils. L1 is called the self-inductance of one coil, L2 is the self-inductance of the other coil, N is the mutual inductance, and the coupling coefficient is expressed as k=N / (L1L2) 1 / 2 .

[0033] Inductive sensing methods can also utilize a combination of one or more coils and / or simple metal structures that are inductively coupled to simplify signal pickup and processing and / or enhance the sensing capabilities of the sensing element. Some examples of such uses of inductive coupling have been reported for magic angle spinning NMR probes or for microprobes used for detection under high pressure in diamond anvil chambers. An example of a sensing element that beneficially utilizes coupling is a Lentz lens inductively coupled to a larger coil. In some embodiments of the present invention, the sensing element of the ultra-compact sensor can also be composed of a stacked combination of microcoils and / or coupled metal structures (e.g., a Lentz lens or the like). The stacked combination can be within a single substrate (i.e., a microchip substrate) or made by combining separate layers that each contain at least one stacked element.

[0034] By "sensing volume" or "sensing region" is meant herein a region of space associated with the excitation and / or sensing of magnetic resonance. Such a region may be defined relative to spin sensitivity. In general, the sensing capability of a magnetic resonance sensor may be expressed as the amount of spins, and therefore the target nuclei and / or electrons in the sample, allowing a signal-to-noise ratio of 3 within a 1 Hz detection bandwidth. Such a value, also referred to as spin sensitivity (ξ), varies in space and is caused by the geometrical properties of the sensing element and the underlying detection mechanism. Thus, given a sensing element and a Cartesian coordinate system (x, y, z), there exists an associated spin sensitivity map ξ(x, y, z). The sensing region size and geometry may be defined relative to such a field of scalar values. By using a grid of space in which all elements have the same volume dV of 1zL to 10zL for each member of the grid, it is possible to relate the respective contribution to the total signal ξ(x i ,y i , z i )dV. In the case of ξmax (the maximum of this set of scalar values), the sensing area should be defined as the union of dV volumes whose signal contributions are individually greater than ξmax / 3. Some embodiments of the present invention relate to devices with individual sensing areas of less than 1 μL.

[0035] A "microcoil" is a coil having a diameter of at least one micrometer (μm) or less than 10 -3A coil or one or more connected rings of meter (mm) size. A microcoil typically comprises a thin material wound or gathered into a spiral, helical or other shape around a center or an imaginary center. The microcoil is defined by the material itself, the winding shape, and the spacing between each winding. A solenoid microcoil is a plurality of spiral wire loops that may or may not be wound around a metal core. A solenoid microcoil generates a magnetic field when an electric current passes through it, and can generate a controlled magnetic field. A solenoid microcoil can generate a uniform magnetic field in a predetermined spatial volume. A "planar" microcoil is a microcoil whose windings are essentially kept in an actual or imaginary plane. Such a microcoil (whose inner diameter size range is typically 0.1 to 0.5 mm (depending on the average size of the sample volume)) is capable of obtaining high-quality NMR spectra with small sample volumes (nL to μL).

[0036] A microcoil array is a collection of microcoils fabricated on a substrate (e.g., a silicon, glass, or polymer substrate). Each microcoil may be associated with or correspond to a sample space through which the microcoil can generate an oscillating magnetic field as part of an NMR or ESR analysis. The sample space may be a space used to hold a sample.

[0037] In one embodiment of the present invention, "body microstructure" means herein a structure placed on top of and / or in the surrounding space of an ultra-compact magnetic resonance sensor. The body microstructure is envisioned to carry a sample (solid and / or liquid), keeping it within the sensing volume. "Enclosure" means herein a container that holds the selected liquid in contact with the sample and the body microstructure. It is obvious that the enclosure and the body microstructure are objects with two different length scale sizes, the former can be called "macroscopic" and the latter can be called "microscopic".

[0038] Some embodiments of the present invention include a main microstructure designed to "only hold" the sample. Such an implementation can consist of a microwell of any shape, where the sample is simply contained within the sensing area. In these embodiments, the user places the sample at the culture microchamber (manually or through an automated system) and can retrieve it once the experiment is over.

[0039] A "microchannel" is a channel having at least one micrometer (μm) or less than 10 -3 A channel, groove or conduit having a size in the order of meters (mm). Although microchannels are usually straight along their length, they may contain angles and curves of varying degrees along their length. Although microchannels usually have rectangular cross-sections, they may also have cross-sections of other shapes, such as circular. Microchannels are generally suitable for fluid communication, such as carrying through biological fluids. Microchannels are generally part of an integrated device (such as a microfluidic device or an integrated circuit) so that the liquid flowing through the microchannel is in a controlled mode and can be analyzed as needed.

[0040] A "microfluidic device" is a device having one or more microchannels. A microfluidic device can be part of an integrated device (e.g., an integrated separation or detection device or an integrated circuit). Fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions, and a variety of buffers and saline.

[0041] Microfluidic devices can be used to obtain many interesting measurements, including fluid mechanical properties, cellular and molecular diffusion coefficients, fluid viscosity, pH, chemical and biological binding coefficients, and enzyme reaction kinetics. Other applications of microfluidic devices include cell and molecular detection and separation, capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, protein sample injection for analysis by mass spectrometry, DNA analysis, cell manipulation, and cell separation.

[0042] As used herein, "magnetic", "magnetic effect" and "magnetism" refer to the phenomenon that one material exerts an attractive or repulsive force on another material. Although in theory all materials are affected by the magnetic effect to some extent, those skilled in the art understand that the magnetic effect or magnetism is only identified because of its detectability in specific circumstances.

[0043] As used herein, a "permanent magnet" is a material that has a magnetic field independent of external influences (e.g., electric current), or any object that is substantially composed of said material. Some metals have magnetic properties when they exist in their natural state (such as ores) due to their unpaired electron spins. These include iron ore (magnetite or lodestone), cobalt, and nickel. "Paramagnetic material" refers to a material that attracts and repels like a normal magnet when subjected to a magnetic field. Paramagnetic materials include aluminum, barium, platinum, and magnesium. "Ferromagnetic material" is a material that can exhibit spontaneous magnetization. Ferromagnetics is one of the strongest forms of magnetism and is the basis of all permanent magnets. Ferromagnetic materials include iron, nickel, and cobalt. "Superparamagnetic material" is a magnetic material that exhibits behavior similar to paramagnetic materials at temperatures below the Curie temperature or the Neel temperature.

[0044] "Solid support" and "support" refer to a material or group of materials having a rigid or semi-rigid surface. In some aspects, at least one surface of the solid support will be substantially flat, although in some aspects, it may be desirable to physically separate synthesis areas for different molecules using, for example, wells, raised areas, pins, etched grooves, etc. In certain aspects, the solid support will take the form of beads, resins, gels, microspheres, or other geometric configurations.

[0045] The terms "chip," "microchip," or "printed circuit board" refer to a small device or substrate that contains components for performing certain functions. A chip includes a substrate made of silicon, glass, metal, polymer, or a combination, and can function as, for example, a microarray, macroarray, and / or integrated circuit. A chip can be a microelectronic device made of semiconductor material and having one or more integrated circuits, or one or more devices.

[0046] A "chip" or "microchip" is typically a portion of a wafer and is made by slicing the wafer. A "chip" or "microchip" may contain many microscopic transistors and other electronic components on a single thin rectangle of silicon, sapphire, germanium, silicon nitride, silicon germanium, or any other semiconductor material. In some embodiments of the invention, microchannels, microfluidic devices, and magnetic tunnel junction sensors may also be integrated into a microchip, as discussed herein.

[0047] As used herein, "maximum dimension" refers to those parameters or measurements required to define the shape and / or size of an object (e.g., height, width, and length). As used herein, the maximum dimension of a two-dimensional object (e.g., a rectangle, polygon, or circle) is the longest straight-line distance between any two points on the object. Thus, the maximum dimension of a circle is its diameter; the maximum dimension of a rectangle is its diagonal and the maximum dimension of a polygon is its longest diagonal. The maximum dimension of a three-dimensional object is the longest straight-line distance between any two points on the object.

[0048] A "microprocessor" is a processor on an integrated circuit (IC) chip. The processor may be one or more processors on one or more IC chips. The chip is typically a silicon chip with thousands of electronic components that serve as the central processing unit (CPU) of a computer or computing device.

[0049] In a first aspect according to the present invention, a method for manufacturing a device is provided, the device comprising a micro-magnetic resonance system operatively connected to a cell culture micro-chamber, the method comprising the step of depositing a thin passivation film or layer on the micro-magnetic resonance system. In some preferred embodiments according to the present invention, the deposition is performed by a deposition process selected from chemical vapor deposition and physical vapor deposition.

[0050] The device of the present invention is a micro NMR or ESR device integrated on a chip, which comprises a substrate, a cell culture microchamber for accommodating a sample operatively connected to a sensing and / or excitation element, wherein the sensing and / or excitation element is capable of generating an excitation magnetic field when passing through at least a portion of the space. For simplicity, the device is also referred to as a "magnetic resonance biological culture microchamber" hereinafter, abbreviated by the acronym μMRBM. Specific materials that can be used as substrates include, but are not limited to, silicon, polystyrene, polydimethylsiloxane (PDMS), glass, chemically functionalized glass, polymer-coated glass, nitrocellulose-coated glass, uncoated glass, quartz, plastic, metal, and ceramic.

[0051] The method of the present invention has been developed to obtain μMRBMs that enable solid samples typically 5 to 1000 μm in size to be brought into close proximity with the sensing element (i.e., as close as 1 nm), either alone or contained in a very small volume of liquid medium (i.e., less than about 1 μL), thereby providing excellent spin sensitivity to the sample under study and a sensing area of ​​less than 1 μL.

[0052] By performing the deposition of layers as a manufacturing step of the device, the device obtained is passivated as a result, uniformly integrated in all its components and functionalizable or otherwise capable of being functionalized as desired and circumstantial.In some preferred embodiments of the method of the invention, a thin passivation film or layer is deposited at least on the sensing element.

[0053] In some preferred embodiments of the method of the present invention, the μMRBM further comprises an active element configured to send an excitation signal to at least one sensing element and / or receive an input signal from the at least one sensing element. In one embodiment, a passivation film or layer is deposited to the active element.

[0054] In one embodiment, the method of the present invention further comprises the step of combining a host microstructure defining a cell culture microchamber volume to the micro magnetic resonance system, thereby creating a cell culture microchamber. Preferably, the cell culture microchamber is located within the sensing and / or excitation region.

[0055] A specific embodiment of the method of the present invention foresees at least the following steps:

[0056] a) providing a micro magnetic resonance system;

[0057] b) providing a main microstructure;

[0058] c) contacting the micro-magnetic resonance system with the main body microstructure, the main body microstructure defining the volume of the cell culture microchamber; and

[0059] d) bonding said micro magnetic resonance system to said host microstructure by a deposition process under conditions allowing to obtain a thin passivation film or layer, thereby creating a cell culture microchamber having a floor consisting of said thin passivation film or layer.

[0060] In a further embodiment, the method further comprises the step of bringing the micro magnetic resonance system into contact with a housing element defining a closed volume.

[0061] It will be obvious to a person skilled in the art that another aspect of the invention relates to a device obtainable by the method of the invention. Specifically, the device comprises a cell culture microchamber with a volume of 0.1 nL to 1 μL coupled to a micro-magnetic resonance system, the cell culture microchamber comprising a bottom plate consisting of a deposited thin passivation film or layer.

[0062] In a preferred embodiment, a miniature magnetic resonance system is available comprising:

[0063] a) at least one sensing element providing a sensing and / or excitation region, and

[0064] b) an active element configured to send an excitation signal to at least one sensing element and to receive an input signal from the at least one sensing element,

[0065] The cell culture microchamber is located in the sensing and / or excitation area.

[0066] The device of the present invention is a versatile and easy-to-use sensor designed for studying and analyzing very small samples by nuclear magnetic resonance (NMR) and electron spin resonance (ESR) techniques. In addition to simply making such spectroscopy tools possible at a volume scale below 1 μL, the present invention also relates to a comprehensive microdevice that allows measurements of biological materials under biocompatible conditions. Such a device allows the user to measure appropriate biochemical parameters of a sample in a systematic manner and in a selected liquid environment under stable temperature conditions and under externally controlled perfusion conditions. The device can be used, for example, to examine the chemical content of a biological sample for the purpose of analysis or diagnosis.

[0067] Preferably, the at least one sensing element and the active element are arranged in an ultra-compact configuration. An "ultra-compact" magnetic resonance device comprises the above two elements stacked vertically or arranged adjacently in a plane. In its implementation, an ultra-compact magnetic resonance sensor comprises two elements in a very small space, the two elements being close enough to each other that the sensing capability is limited only by the sensing element and not by the interconnection between the two elements. In such an implementation, the sensor is locally operated, thereby reducing unwanted signals from materials placed around, below and above the ultra-compact device.

[0068] In another embodiment according to the present invention, the device comprises a permanent magnet or an electromagnet capable of generating a static magnetic field when passing through at least a portion of the space. The permanent magnet or electromagnet generates a static magnetic field when passing through at least a portion of the space for accommodating the sample. Materials suitable for use as permanent magnets or electromagnets include permanent magnetic materials, ferromagnetic materials, paramagnetic materials, and non-magnetic metals. When ferromagnetic materials are used for magnets, an external magnetic field is used to magnetize the material. In addition, when ferromagnetic materials or non-magnetic materials are used for magnets, an electric current is applied to the material to generate an electromagnet. In one embodiment of the present invention, the magnet comprises one or more of iron, nickel, cobalt, rare earth materials (e.g., neodymium, copper, aluminum) and mixtures thereof. In some embodiments, the magnet can generate a static magnetic field intensity of about 0.01 Tesla (T) to about 30T, such as about 0.01T to about 10T, about 0.01T to about 7T, or about 0.01T to about 5T.

[0069] Preferably, the thin passivation film or layer is thinner than one third of the largest linear dimension of the sensing element and in some embodiments comprises a material selected from the list comprising: resist, photoresist, parylene C, SiO2, Si x N y , ceramics and polymer derived ceramics. The ratio between the thin passivation film and the largest linear dimension of the sensing element is an important parameter to set, since the sensitivity decreases rapidly with movement away from the sensing element surface and it decreases in direct proportion to a power function of the largest linear dimension of the sensing element.

[0070] For example, in order to have optimal spin sensitivity in the sensing area of ​​the device, and assuming that there are microcoils with a maximum linear dimension of about 180 μm in the sensing or excitation element, the passivation film will preferably have a thickness of less than 15 μm for embryo analysis (dimension of about 100 μm). In the case of a sensing or excitation element with a maximum linear dimension of 500 μm (for stem cell-derived tissues with a dimension of about 300 to 600 μm), a passivation film thickness of up to about 40 μm may be suitable. For samples with a dimension of about 1 mm, such as organoids or so-called cancer droplets, a passivation film thickness of up to about 100 μm will be very suitable.

[0071] Some embodiments of the present invention contemplate magnetic resonance bio-culture microchambers based on optomagnetic sensors as sensing elements. It is now known that optomagnetic phenomena can directly detect the sample magnetization dynamics that are typical features of magnetic resonance experiments. An example of such a sensing scheme is the sensing scheme of nitrogen vacancy defects in a diamond structure, where the optical readout from the sensing element is based on excitation and detection with light, and in some implementations, the excitation and detection with light is combined with microwave excitation of the Zeeman level of the electrons present in the defect. Such diamond structures can also be considered the basis of ultra-compact magnetic resonance tools and are often referred to as "color centers."

[0072] Reference Figure 1 , shows a schematic embodiment of a complete magnetic resonance device ("magnetic resonance biological culture microchamber" or "μMRBM") 100 containing an NMR microsystem according to the present invention. In the depicted embodiment, the device 100 is inserted into a socket 1000 and is inserted into a permanent magnet or electromagnet 5000 for use. The characteristics of the magnet 5000 can vary depending on the method used to generate the static magnetic field B. The permanent magnet 5000 can generate a magnetic field B with an intensity of up to 1 T, and when the Halbach configuration is used, up to several T above this value. For high magnetic fields, i.e. 5 T and above 5 T, superconducting coils injected with high current density are generally used. The connection between the socket 1000 and the printed circuit board on which the μMRBM 100 is carried is achieved by a male-female electrical connector. A multi-signal cable 4000 connects the socket to a console 3000 containing known electronic devices and allows the user to control experimental parameters and collect data through a computer system 2000.

[0073] Reference Figure 2 a, shows a schematic top view of two embodiments of the geometry of the ultra-compact sensor 101. In the embodiment shown on the left, the sensor 101 includes a sensing and / or exciting element 300 that is in close proximity and adjacent in the plane to the active element 200, and a plurality of spacers 301, while in the embodiment on the right, the sensing and / or exciting element 300 is surrounded by the active element 200. Figure 2 b shows a 3D rendering of the same two embodiments, further showing the bonding wires 302 connecting the pad 301 to the printed circuit board 400 .

[0074] Figure 3 One embodiment of a schematic view of a μMRBM 100 is illustrated in FIG. The basic μMRBM 100 comprises four main components: an ultra-compact magnetic resonance sensor 101, a main microstructure 500, a cover 700 and a passivation bonding layer 800. Figure 3In the embodiments shown in a and c, a solid sample 600 (e.g., a biological sample of an embryo) is placed in the main microstructure 500, in the culture microchamber 502, corresponding to the sensing and / or excitation element 300, so as to sense the magnetic field in the sensing and / or excitation area. By including a culture medium 601 in a closed structure 700, the sample 600 is maintained under appropriate culture conditions. Figure 3 In the embodiment shown in b, the closure cap 701 seals the closure structure 700, thereby ensuring sterility and preventing leakage of the medium.

[0075] Some embodiments of the present invention include additional features to address further needs such as: the need to perform measurements at a controlled temperature, the need to place the μMRBM vertically, the need to perfuse the μMRBM, the need to capture biological samples at the sensing area, the need to control other properties of the culture medium, the need to perform spin operations using auxiliary coils outside the ultra-compact sensor, etc.

[0076] In some embodiments, the body microstructure 500 can be operatively connected to a microfluidic channel so as to enable fluid connections to be made, for example, to flush a liquid or medium 601 to or from the body microstructure 500. In some embodiments, the flushing liquid also serves as a carrier for driving the sample 600 to or from the capture site. In these embodiments of the present invention, the sample 600 is retained in the culture microchamber 502 while being confined in a direction perpendicular to the surface of the ultra-compact device, particularly within the sensing region defined by the sensing element 300. Such features can be used, for example, to use the μM RBM 100 in a perpendicular magnetic field, where the gravitational effects and the manipulation required to place the μM RBM 100 in the magnetic field can cause the sample 600 to detach from the culture microchamber 502 and therefore the sensing region. These embodiments of the present invention can also be used to address the needs of array implementations in which multiple samples are analyzed simultaneously and / or in large quantities in a pipeline. In embodiments thereof, placement of a large number of samples and their retrieval may be addressed in an automated fashion and / or in a single flushing operation.

[0077] Some embodiments of the present invention include a cover (501; Figure 3b) of the main microstructure 500. "Top cover of the main microstructure" means in this article an element on the top of the main microstructure 500, which can be used to confine the sample 600 carried in a direction perpendicular to the surface of the ultra-compact device 100. Such a feature is essential for, for example, using the μMRBM 100 in a vertical magnetic field, in which the gravitational effects and the manipulation required to place the μMRBM in the magnetic field can cause the sample to escape from the culture microchamber 502 and therefore the sensing area. In these embodiments, the user is able to manually and / or automatically operate the top cover 501 of the main microstructure 500 to enter the culture microchamber 502 for manual and / or automatic sample 600 placement and retrieval.

[0078] Some embodiments of the present invention include a top cover 501, which may have different functions relative to the liquid medium 601 contained in the housing 700 and surrounding the sample 600 in the main microstructure 500. The top cover 501 may allow the liquid to flow freely between the culture microchamber 502 and the housing. Such a top cover may be implemented, for example, by having a perforated structure as the top cover. The top cover may also include a semi-permeable portion, such as Figure 3 b, it can selectively prevent molecular substances from flowing outward from the culture microchamber 502 to the liquid reservoir contained in the housing 700 and / or produced by the sample 600 (e.g., produced by chemical reactions or metabolism of the biological sample). Similarly, the semi-permeable portion can also prevent molecular substances consumed by the sample (e.g., consumed by chemical reactions or metabolism of the biological sample) from flowing inward in the culture microchamber 502. The top cover can prevent any liquid exchange between the culture microchamber 502 and the housing 700.

[0079] Some embodiments of the present invention include a top cover 501 based on a "bistable mechanism". A "bistable mechanism" is a mechanism in which the potential energy of the system has two local minima separated by a maximum. The minimum is the two possible equilibrium positions of the system, which can be called open / closed or on / off. The maximum is used as a barrier between the two positions. Switching between the two equilibrium positions requires applying sufficient, controlled external force. The force can be provided by several methods including: gravity redirected by μMRBM 100, mechanical force locally applied to the top cover 501, piezoelectric system, electrophoresis system, optomechanical system acting on the top cover 501, magnetic system, pneumatic system or a suitable combination thereof. Some embodiments of the present invention also include a "multi-stable mechanism", in which there are more than one local minima of potential energy. An example of such a multi-stable mechanism is a ratchet system.

[0080] Some embodiments of the present invention include a top cover 501 based on a "pivot mechanism". A "pivot mechanism" is a mechanism in which the top cover can rotate freely around a pivot point placed on the main microstructure 500. An example is a mechanism in which the direction of rotation is parallel to the top surface of the main microstructure 500. In such a configuration, the rotation angle range of the top cover can be infinite or limited, for example by appropriately placing protrusions on the surface of the ultra-compact device 100. Another example of a pivot mechanism is a mechanism in which the top cover 501 rotates around a joint parallel to the top surface of the main microstructure 500. In such a configuration, the top cover 501 will serve as a hatch for the culture microchamber 502.

[0081] Some embodiments of the present invention include a top cover 501 based on a "sliding door mechanism". Such a mechanism is one in which the top cover 501 slides in a slot incorporated into the main microstructure 500. The sliding direction and range are defined by the slot. An example is one in which the slot is a groove in the main microstructure that matches a complementary protrusion on the top cover 501.

[0082] According to one embodiment of the present invention, the main microstructure 500 and the top cover 501 can be manufactured by two-photon photopolymerization using laser direct writing. In the case of a "bistable mechanism", the main microstructure 500 and the top cover 501 are manufactured as a single unit that remains intact during use. In another embodiment (e.g., a "pivot mechanism" or a "sliding door mechanism"), the main microstructure 500 and the top cover 501 can be manufactured as an integral structure, wherein the two are held together by a connector that is removed before use, thereby obtaining two separate objects. Alternatively, the two objects can be manufactured separately and then combined before use.

[0083] Some embodiments of the present invention include a main microstructure 500 with a sealed culture microchamber 502, in which a substance (e.g., water or rubber) is enclosed in the culture microchamber during manufacture. Such a closed substance can be manufactured by any sealing mechanism, for example, by a bonding technique, wherein the microstructure is first manufactured with the top open, and then closed by bonding a covering layer. It is also possible to use materials (polymers, glass, semiconductors, ceramics) with a high degree of compatibility with magnetic resonance experiments of liquids and biological samples to be combined between microstructures. These are all recognized methods for the situation of each specific material to properly seal the channel to avoid liquid leakage.

[0084] According to one embodiment of the present invention, the main microstructure 500 can be manufactured by a microfabrication technique based on a sequence involving material coating, photolithography, development and etching. In particular, the covered main microstructure can be manufactured by surface micromachining using a sacrificial layer method, in which the buried structural layer is removed by an etchant or solvent through a sacrificial removal step. For example, high aspect ratio polymer lithography (such as SU-8, Ormocers, etc.) is a transparent thick resist that meets the microstructure requirements.

[0085] The geometrical characteristics and properties of the materials used to realize the bulk microstructure are considered in the design, as they significantly affect the performance of the device. Those skilled in the art do know that both the geometrical characteristics and the magnetic susceptibility characteristics are extremely important parameters, as they are directly related to the magnetic field distortion in the sensing region and, therefore, to the spectral line broadening. The magnetic susceptibility of the material is denoted as χv and is given by the relation M = χ v H0 definition, where M is the magnetization of the material when it is incorporated into an external magnetic field strength H0. In this definition, χv is a dimensionless quantity and is related to the ability of the material to produce magnetization when subjected to a magnetic field.

[0086] Since the μMRBM according to the invention is envisaged, among other applications, for the analysis of samples immersed in a culture medium, it is assumed that a liquid medium with a magnetic susceptibility similar to that of water (H2O) will be present in the sensing region. Such a liquid will be in direct contact with the edges of the host microstructure and will therefore produce a surface in which a mismatch in magnetic susceptibility exists. χ1 and χ2 are called the magnetic susceptibilities of the mismatched surfaces and will result in a magnetic field distortion that is directly proportional to (χ1-χ2)B0 sin(θ), where θ is the angle between the external static field and the mismatched surface.

[0087] Therefore, the main microstructure 500 of some embodiments of the present invention is made of a material that is highly compatible with magnetic resonance experiments of liquids and biological samples. Such materials are characterized by a magnetic susceptibility that is not more than 10 ppm of xH2O different from the magnetic susceptibility of water. Such materials include, among others: silicone, photoresist, most polymers (e.g., acrylics), epoxy resins, gels (e.g., alginate and agarose-based gels), glass, polyurethane, Teflon, ceramics.

[0088] In the μMRBM 100 according to the present invention, an essential element is Figure 3The passivation bonding layer 800 shown in . Such a layer 800 has multiple roles: as a bonding element, as a passivation layer and as a functionalized layer. The bonding is between the main microstructure 500 and the ultra-compact sensor. Such a bonding role is crucial to give the necessary robustness, which makes the μMRBM100 user-friendly and easily accepted in daily practice. The role of passivation is crucial to prevent electrical connection between the sensor and the medium. Without passivation, the μMRBM will face problems in its electrical functionality.

[0089] According to a specific embodiment of the invention, the passivating bonding layer can be manufactured by physical vapor deposition (PVD) techniques, thermal evaporation or electron beam evaporation in a vacuum chamber, or by sputtering in a plasma chamber. Both methods are well established for the deposition of thin dielectric films such as parylene, SiO2, SiN, etc. In some variations, the coated film is conformal and covers not only the top surface, but also the side walls and the portion on the specimen that is in the shadow of the material source. Spraying of ceramics of ceramic or polymer origin is a possible method to obtain the passivating bonding layer.

[0090] In some embodiments of the present invention, the passivating binding layer 800 is used as a substrate suitable for both cell growth and exposure to a specific medium by surface treatment. The functionalized layer 800 allows the user to customize the surface properties according to needs and circumstances (such as the application sought and the sample to be analyzed) to expand the scope of application. For example, poly-chloro-p-xylene (commonly known as parylene C) is a suitable material for the passivating binding layer 800 in μM RBM 100 because it is known to have high-quality packaging, electrolyte barrier characteristics, chemical inertness, and biocompatibility. It is also known to those skilled in the art that the surface of a material (such as parylene C, SiO2 or other candidate passivating binding layer) can be treated to change the hydrophilic properties and improve the interaction with the growing cells contacting the substrate. Some embodiments of the present invention include μM RBM 100, which has a surface-treated passivating binding layer 800 to improve its biocompatibility and hydrophilic properties. It is known that the parylene C passivating binding layer 800 used in a preferred embodiment of the present invention exhibits properties that compare well with standard tissue culture substrates once its surface is properly treated.

[0091] In some embodiments of the present invention, μMRBM 100 undergoes oxygen plasma treatment, UV radiation, silanization process, additional coating operations, on-demand inkjet printing, local PVD through a shadow mask, polymer grafting. All of these treatments are contemplated for use alone or in combination through a multi-step approach. In some embodiments of the present invention, only one surface treatment is applied to the entire exposed surface. In some embodiments of the present invention, only one surface treatment is performed on a target area of ​​the exposed passivating bonding layer 800. In some embodiments of the present invention, there is a combination of surface treatment operations that individually target the entire exposed surface, a single or multiple sub-areas of the exposed passivating bonding layer 800. In some embodiments of the present invention, the same surface treatment targeting different areas of the passivating bonding layer 800 or the entire exposed surface is applied in multiple steps.

[0092] In some embodiments, the device of the present invention includes an ambient temperature sensor within the housing 700 to measure the temperature of the surrounding liquid / culture medium. In addition, in some further embodiments according to the present invention, the μMRBM 100 may include a heating element and / or a cooling element intended to control the temperature of the surrounding liquid / culture medium.

[0093] In both NMR and ESR, manipulation of the sample magnetization and / or the magnetic field in which the sample is trapped provides a powerful method for extracting information during an experiment. One example is the so-called CPMG (Carr-Purcell-Meiboom-Gill) sequence, in which the sample magnetization is flipped in space with the purpose of measuring the relaxation properties of the NMR signal. Another example is the WATERGATE sequence (Water Peak Suppression by Gradient Clipping Excitation), in which pulsed field gradients are added to the static field and combined with selective excitation pulses, allowing the water signal to be suppressed. Magnetic resonance imaging gives another example, in which pulsed field gradients are the basis of the image reconstruction process.

[0094] In some embodiments, the μMRBM 100 of the present invention comprises one or more inductors, also referred to herein as “auxiliary coils” 303 / 305 , which are configured to introduce manipulation of sample magnetization (beyond manipulation performed with the excitation / detection elements) and / or manipulation of external static fields (e.g., via field gradients and / or spin flipping; Figure 4 and 5). Such an inductor 303 / 305 may also be used to sense or improve the sensing power of the μMRBM 100. Such an auxiliary coil 303 / 305 is: connected to a board 400 carrying the μMRBM 100; connected to leads separated from the μMRBM printed circuit board 400; shortened for operation via inductive coupling; connected to a structure that is part of an insert that directly supports or does not directly support the μMRBM 100; connected to a structure supported by and / or guided only by a magnet hole / opening; connected to a microchip pad; connected to microchip electronics; connected to microelectronics co-integrated with an ultra-compact sensor. Auxiliary coils may also be classified as "external" or "internal" coils.

[0095] like Figure 4 As shown in , some embodiments of the present invention include an "external auxiliary coil" 303, which is intended to manipulate the sample magnetization and / or manipulate the external static field by circulating an electric current 304. The external auxiliary coil 303 is an inductor of any geometry capable of generating magnetic disturbances in the sensing area of ​​the μMRBM 100, and whose metal windings are such that the current passing through the coil does not flow through metal belonging to the same microchip substrate containing the ultra-compact sensor 101. The external auxiliary coil 303 can be included in a microchip or microstructure other than the microchip or microstructure in which the ultra-compact sensor 101 is integrated. The external auxiliary coil can be implemented in the main microstructure 500 defining the μMRBM 100. Figure 4 Two basic examples of external auxiliary coils 303 that complement the ultra-compact sensor 101 are shown. One way to add gradient and / or excitation coils is to physically place them around the μMRBM 100: this strategy can be used to recycle the standard pulsed gradient coils used in standard NMR probes ( Figure 4 a).

[0096] Figure 4 b also shows the possibility of using a single-sided coil placed in close proximity to the μMRBM 100. Such a coil can be used to achieve both pulse gradients or excitation pulses. For example, its use for excitation pulse generation can be advantageous where magnetic field homogeneity is critical or in heteronuclear excitation experiments. The external auxiliary coil 303 can have any geometry and size. The external auxiliary coil 303 can be placed around the μMRBM housing 700 (thus not in contact with the liquid medium 601), inside the housing 700 (thus in contact with the liquid medium 601), or a combination of the two.

[0097] Some embodiments of the present invention comprise an "internal auxiliary coil" 305, which is intended to operate on sample magnetization and / or on external static fields. An internal auxiliary coil is an inductor of any geometry capable of generating magnetic perturbations in the sensing region of the μMRBM 100, whose metal windings cause the current through such an inductor (at least part of its total path) to flow through metal belonging to the same microchip substrate containing the ultra-compact sensor 101 (a bonding pad of this type can be considered). Figure 5 Two basic examples of internal auxiliary coils 305 are illustrated. Figure 5 In a, wire bonds and on-chip metallization form an inductor that can be used for field manipulation within the sensing region. The internal auxiliary coil can be made in any geometry and have any size. Figure 5 In b, the case of an auxiliary coil 305 that is strongly coupled to the sensing element 300 and is realized entirely with metal layers available on the microchip carrying the ultra-compact sensor 101 is shown. In the same way, gradient coils acting locally in the sensing area can be realized. The internal auxiliary coil 305 is at least partially placed inside the housing 700 (and therefore in contact with the liquid medium 601). The internal auxiliary coil 305 can also be used to improve or supplement the sensing more effectively than an external auxiliary coil: Some embodiments of the present invention include a μMRBM 100 with multiple induction coils targeting the same volume space.

[0098] Some embodiments of the present invention foresee array μMRBMs, where multiple μMRBMs 100 can operate in the same static magnetic field ( Figure 6). The array μMRBM can be implemented as a collection of separate μMRBMs 100 placed on the same printed circuit board 400 or on separate circuit boards connected to each other in a modular manner. The array μMRBM includes multiple excitation / detection elements 300 that can be connected to: a single integrated electronic system implemented on a single microchip substrate 101, a single integrated electronic system implemented on multiple microchip substrates 101, independent electronic systems implemented on a single microchip substrate 101, independent electronic systems implemented on separate microchip substrates 101. According to the definition of μMRBM, the ultra-compact array sensor 101 is implemented together with a main microstructure 500, a passivation bonding layer 800 and an external cover 700. The main microstructure 500 can be composed of: a single microstructure connected to all excitation and / or detection elements 300, multiple and separate microstructures 500 connected to a single excitation and / or detection element 300, or multiple and separate microstructures 500 connected to more than one excitation and / or detection element 300. The housing 700 in the array μMRBM may be a housing containing the entire array, or a housing separated into a plurality of independent housings 700 containing one μMRBM or an array μMRBM. All of the above configurations and combinations of these configurations define an array μMRBM.

[0099] Figure 6 a shows an array μMRBM in which individual μMRBMs 100 are placed on the same printed circuit board 400 . Figure 6 b shows an array μMRBM comprising multiple ultra-compact sensors 101 and a single main microstructure 500 that independently targets each excitation and / or detection area. Multiple μMRBMs 100 can be used in parallel using the same experimental parameters to improve throughput and therefore statistics. Alternatively, different experiments can be performed simultaneously using individual μMRBMs 100 in the array, such as: one-dimensional spectroscopy, two-dimensional spectroscopy (correlation spectroscopy, J-spectroscopy, exchange spectroscopy, etc.), measurement of relaxation times T1 and T2. In addition, a subset of μMRBMs can be used without samples and for background measurements. Similarly, for some embodiments of the array μMRBM, auxiliary coils 303 / 305 designed to operate on sample magnetization and / or on external static fields can be envisioned.

[0100] References

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[0102] 2.Olson,D.L.,et al.,High-Resolution Microcoil H-1-Nmr for Mass-Limited,Nanoliter-Volume Samples.Science,1995.270(5244):p.1967.

[0103] 3.Spengler,N.,et al.,Micro-fabricated Helmholtz coil featuringdisposable microfluidic sample inserts for applications in nuclear magneticresonance.Journal of Micromechanics and Microengineering,2014.24(3).

[0104] 4.Grisi,M.,G.Gualco,and G.Boero,A broadband single-chip transceiverfor multi-nuclear NMR probes.Review of Scientific lnstruments.2015.86(4).

[0105] 5.Ha,D.,et al.,Scalable NMR spectroscopy with semiconductorchips.Proceedings of the National Academy of Sciences,2014.

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Claims

1. A micro MR device (100) for analyzing biological samples having a size less than about 1000 μm, comprising: A miniature magnetic resonance system (101), the miniature magnetic resonance system (101) comprising: a) at least one sensing element (300) providing a sensing and / or excitation region, and b) an active element (200) configured to send an excitation signal to the at least one sensing element (300) and / or receive an input signal from the at least one sensing element (300), and contains electronic devices; a housing (700) defining an enclosed volume and containing a liquid; and A main microstructure (500) defining a cell culture chamber (502) having a volume of 0.1 nL to 1 μL; Features The cell culture chamber (502) is contained in the housing (700) and is in contact with the liquid in the housing (700); The device comprises a passivating bonding layer (800) covering at least a portion of the system (101) and the chamber (502), the passivating bonding layer (800) serving as an isolating shielding material to prevent physical, chemical and / or electrical connection between the electronic components and the liquid medium; and The sensing element (300) and the active element (200) are co-integrated on the same substrate (400), adjacent in a plane or stacked vertically, and the culture chamber (502) is located on top of the sensing element (300).

2. The device according to claim 1, comprising a sensing and / or excitation area defined by the sensing element (300), wherein the chamber (502) is located within the sensing and / or excitation area.

3. The device of claim 2, wherein the sensing area is less than 1 μL.

4. The device according to any of the preceding claims, wherein the passivating bonding layer (800) has a thickness of 1 nm to 100 μm.

5. The device according to any one of claims 1 to 3, wherein the sensing element (300) comprises a microcoil having an inner diameter of 0.1 to 0.5 mm.

6. The device according to any one of claims 1 to 3, wherein the cultivation chamber (502) has a bottom plate consisting of the passivating bonding layer (800).

7. The device according to any one of claims 1 to 3, wherein the layer (800) comprises a thin film of a deposited material selected from the list comprising: resist, photoresist, parylene C, SiO2, Si x N y , ceramics and polymer derived ceramics.

8. The device according to any one of claims 1 to 3, further comprising a top cover (501) located on top of the main microstructure (500), wherein the top cover (501) is configured to confine the biological sample (600) within the culture chamber (502).

9. The device according to any one of claims 1 to 3, further comprising a permanent magnet or an electromagnet (5000) capable of generating a static magnetic field.

10. A method for manufacturing a micro MR device (100) for analyzing biological samples with a size less than about 1000 μm as claimed in any one of claims 1 to 8, comprising the following steps: a) providing a miniature magnetic resonance system (101), wherein the miniature magnetic resonance system (101) comprises: a1) at least one sensing element (300) providing a sensing and / or excitation area, and b1) an active element (200) configured to send an excitation signal to the at least one sensor element (300) and / or receive an input signal from the at least one sensor element (300), and contains electronic devices; b) providing a main microstructure (500) defining a cell culture chamber (502) having a volume of 0.1 nL to 1 μL; c) bringing the micro magnetic resonance system (101) into contact with the microstructure (500); and d) bonding the system (101) to the microstructure (500) by a deposition process under conditions allowing obtaining the passivating bonding layer (800), the passivating bonding layer (800) acting as an isolating shielding material preventing physical, chemical and / or electrical connection between the electronic components and the liquid medium and covering at least a portion of the system (101) and the cell culture chamber (502), thereby creating a cell culture chamber (502) having a floor consisting of the layer (800), The method further comprises the step of bringing the system (101) into contact with a housing (700) defining a closed volume, The sensing element (300) and the active element (200) are co-integrated on the same substrate (400), adjacent in a plane or stacked vertically, and the culture chamber (502) is located on top of the sensing element (300). The method of claim 10 , wherein the deposition process is selected from chemical vapor deposition and physical vapor deposition.

12. The method according to any one of claims 10 to 11, wherein the thickness of the passivation bonding layer (800) is 1 nm to 100 μm.

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