CN112034163A

CN112034163A - Method, device and kit for detecting biological small molecules

Info

Publication number: CN112034163A
Application number: CN202010485991.2A
Authority: CN
Inventors: 段学欣
Original assignee: Anhang Biotechnology Co ltd
Current assignee: Anhang Biotechnology Co ltd
Priority date: 2019-06-03
Filing date: 2020-06-01
Publication date: 2020-12-04

Abstract

The invention provides a method for directly detecting biological small molecules in a solution by using a system for generating bulk acoustic waves by using an ultrahigh frequency resonator. The invention also provides a device or a device for detecting the biological small molecules based on the method. The method and the device provided by the invention are suitable for clinical detection, in particular to miniaturized instant detection.

Description

Method, device and kit for detecting biological small molecules

The present application claims priority from the following chinese patent applications: the invention is a method and a device for detecting small biological molecules and a kit, which are filed in 2019, 6, 3 and have the application number of 201910476691.5 and the name of biological small molecular, and the whole content of the method and the device is incorporated into the application by reference.

Technical Field

The present invention relates to the field of cell research methodologies and medical devices. In particular, the present invention relates to an apparatus for separating and detecting biomolecules and a method for separating and detecting biological substances using the same.

Background

The purification and detection of biomolecules, such as metabolites and other small molecules, as well as proteins and nucleic acids, is a central issue of great interest in many fundamental fields of biological mechanism research, medicine and disease diagnosis, and medical instrument development.

Various physical fields are used in the prior art to focus target biomolecules at a specific local area. For example, the nano-sized particle material is used as a carrier, the biological target molecules (blood, saliva, urine, etc.) in the complex biological sample are specifically purified, and then the nano-sized particle carrier is operated, so that the operation of the target biological molecules is realized. For example, the capture and confinement of nanoparticles and molecules to achieve local intra-area aggregation has long been a sought-after goal in the fields of ultra-high sensitivity biosensors, novel drug delivery methods, and the like. High speed centrifugation is used to separate nanoparticles of different sizes; dielectrophoresis techniques have also been used to capture the accumulated and assembled nanoparticles; both magnetic and optical tweezers techniques are used to manipulate nanoparticles and biomolecules. The existing molecular enrichment technology comprises an electrophoresis technology applying high voltage, a molecular enrichment assisted by a nano flow channel, a molecular screening realized by a nano filtering membrane and the like.

However, these techniques suffer from the disadvantages of high energy, low flux and susceptibility to blocking, and require very large field intensity distributions to be generated locally, which has major limitations for molecular manipulation. At the same time, the specificity of manipulating molecules directly using physical fields is poor.

The portability of detection equipment is a great trend of the development of modern scientific instruments. Portable biosensing devices have wider applications such as home health detection, real-time detection of human health indicators, and diagnostics in remote areas where medical resources are scarce. Generally speaking, the portable sensor has the characteristics of small volume, simple operation, rapidness, reliability, relatively low price and the like. Accordingly, only the biosensor technology or the biosensor method conforming to the above characteristics is expected to be developed as a portable biosensor at first.

Therefore, there is a need for a more efficient and convenient method and system for the enrichment, isolation and detection of biomolecules.

Disclosure of Invention

The invention provides a novel method for separating, enriching and detecting biological substances by using a system for generating bulk acoustic waves by using an ultrahigh frequency resonator. The invention also provides a separation and detection device for separating, enriching and detecting biological substances by the method. The separation and detection method provided by the invention has the characteristics of low material requirement, simple operation, short detection time period, less required samples and high sensitivity.

The method and the device provided by the invention provide a powerful tool for detecting the biomolecules, particularly miniaturized instant detection.

Specifically, the present invention provides a method for detecting a target substance in a sample, the method comprising:

processing a sample containing a target substance in an apparatus comprising; a container for holding a solution; one or more UHF bulk acoustic resonators disposed on a wall (e.g., bottom) of the vessel, the UHF bulk acoustic resonators capable of generating bulk acoustic waves having a frequency of about 0.5-50 GHz; the top of the UHF bulk acoustic wave resonator is provided with a ligand which is combined with a target substance;

and the ultrahigh frequency resonator emits a bulk acoustic wave, so that the solution in the bulk acoustic wave region generates a rotational flow, and when the target substance exists in the solution, the target substance enters the rotational flow, is collected by the rotational flow, and is contacted and combined with the ligand at the top of the ultrahigh frequency bulk acoustic wave resonator.

In one aspect of the invention, the target substance of the method is a small biological molecule substance, such as a peptide, polypeptide, protein, lipoprotein, glycoprotein, nucleic acid (DNA, RNA, PNA, aptamer), and nucleic acid precursor (nucleosides and nucleotides), polysaccharide, lipid, and the like. The protein may include, among others, cytokines, vitamins, surface receptors, haptens, antigens, antibodies, enzymes, growth factors, recombinant proteins, toxins, and fragments and combinations thereof. The small biological molecule species are typically on the order of nanometers or smaller, e.g., less than 100 nm.

After the target substance is contacted and bound to the ligand at the top of the UHF bulk acoustic wave resonator, the target substance can be detected by detecting the detectable label carried by the target substance. Such labels include, without limitation, fluorescence or other forms of luminescence (e.g., chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallography, thermoluminescence, sonoluminescence, phosphorescence, photoluminescence, and the like), enzymatic reactions, radioactivity, and the like.

The method of the present invention can be used to detect a biological substance in a sample. The sample may be a culture solution containing a target substance or a suspension thereof or a supernatant thereof, a buffer solution, for example, a culture solution of various viruses, bacteria or cells or a suspension thereof or a supernatant thereof. There are various body fluids, e.g., of various animals, such as humans, including blood, interstitial fluid, extracellular fluid, lymphatic fluid, cerebrospinal fluid, aqueous humor, urine, sweat, and the like.

In one aspect of the present invention, the target biological substance can be selectively bound to the device surface by the specific binding of the binding partner to the target biological substance after the top surface of the uhf bulk acoustic resonator acts as a solid support with the corresponding binding ligand (binding ligand) for the target biological substance thereon. The uhf bulk acoustic wave resonator is located at the bottom of the vessel of the apparatus of the present invention, so the top of the uhf bulk acoustic wave resonator is also typically part of the bottom surface of the vessel.

"binding partner" refers to any biomolecule or other organic molecule capable of binding to or interacting with (particularly specific binding to or interacting with) another biomolecule. Such binding or interaction may be referred to as "ligand" binding or interaction. For example, but not limited to, antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer binding or interaction.

Thus, the inventive uhf bulk acoustic wave resonator has a ligand on the top surface that (specifically) binds to a target substance, which target substance and the ligand are each an antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer. Suitable ligands may be selected according to the desired use of the vector of the invention.

The ligands can be bound to the device surface by methods known in the art. Direct binding can be achieved, for example, by reductive amination or by reacting a nucleophilic group on a binding ligand with an activated ester side chain, such as an N-hydroxysuccinimide activated ester, on a polymer on the device surface. As another example, amine groups and carboxylic acid groups on ligands and polymers can be linked by conventional peptide-forming chemistry, such as using carbodiimides.

The target substance of the method provided by the present invention is a biological substance, and is generally a molecule having a specific structure, such as a peptide, a polypeptide, a protein, a lipoprotein, a glycoprotein, a nucleic acid (DNA, RNA, PNA, an aptamer (aptamer), etc.), a nucleic acid precursor (nucleoside and nucleotide), a polysaccharide, a lipid, such as a lipid vesicle, etc. The protein may include, among others, cytokines, vitamins, surface receptors, haptens, antigens, antibodies, enzymes, growth factors, recombinant proteins, toxins, and fragments and combinations thereof. The biological material typically has a size on the order of nanometers or less, e.g., less than 100 nm.

The method of the invention also comprises detecting the separated target substance, for example by detecting a characteristic or marker signal of the target substance itself bound on top of the uhf bulk acoustic wave resonator. The detection of biological substances can be carried out by various known methods, in particular visual detection. Specific ligands with fluorescent or other forms of luminescence (e.g., chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallography, thermoluminescence, sonoluminescence, phosphorescence, photoluminescence, and the like), enzymatic reactions, radioactivity, and the like may be used to recognize the target substance. For example, when the target substance is a protein or an enzyme, the protein or the enzyme can be specifically recognized using an antibody labeled with a fluorescent or radioisotope, and the target substance can be detected by detecting the fluorescent or radioisotope. Preferably, the detection is carried out by fluorescence. The target substance may be labeled with a detectable fluorophore, and the emitted fluorescent signal is detected by the optical detection unit.

The method of the invention utilizes the ultra-high frequency bulk acoustic wave resonator to generate ultra-high frequency bulk acoustic waves in the solution, standing waves are not generated basically in the solution, the volume force generated by the attenuation of the acoustic waves into the fluid causes acoustic jet flow to appear in the flowing solution, and the fluid with the same flux is caused to flow back to generate vortex. The multiple continuous vortexes generated by the UHF resonator form an acoustic fluid vortex channel. The method of the present invention presents the interaction of the various fluid streams and the particles therein in the vortex created in the solution. Particles that have passed through the vortex of the bulk acoustic wave region are subjected to a combination of fluid drag forces (Stokes drag forces) and acoustic radiation forces (acoustic radiation forces) caused by acoustic attenuation. Because the size of the particles in the invention is in nanometer level, the force is mainly the fluid dragging force in the vortex, and the fluid drags around the center of the vortex in the vortex, and one end of the motion track of the fluid drags and contacts the top of the UHF resonator. Thus, in the presence of the UHF bulk acoustic wave resonator creating a vortex in the solution, the particles in the solution will continue to enter the vortex and will continue to approach and contact the top of the UHF resonator. The applicant has unexpectedly found that particles moving in said swirling flow, when approaching a stationary counterpart ligand, are still able to undergo effective binding.

In the method of the invention, the UHF bulk acoustic wave resonator can generate high-frequency (about 0.5-50GHz) vibration to induce bulk acoustic waves with corresponding frequencies in the solution. In one aspect of the present invention, the ultra high frequency bulk acoustic resonator is a Film Bulk Acoustic Resonator (FBAR) or a solid state fabricated resonator (SMR), preferably a solid state fabricated resonator. In yet another aspect of the present invention, the uhf bulk acoustic resonator is an acoustic resonator of a thickness extensional vibration mode, and the thin film layer of piezoelectric material is grown in a vertical direction, and the vibration is excited by coupling a vertical electric field with a d33 piezoelectric coefficient. The ultrahigh frequency bulk acoustic wave resonator adopted by the invention can generate localized acoustic current at the interface of the device and liquid without the help of a coupling medium or a structure. The acoustic wave resonator comprises an acoustic wave reflecting layer, a bottom electrode layer, a piezoelectric layer and a top electrode layer which are sequentially arranged from bottom to top. The overlapped area of the bottom electrode layer, the piezoelectric layer, the top electrode layer and the acoustic wave reflecting layer forms a bulk acoustic wave generating area. The top surface of the ultrahigh frequency bulk acoustic wave resonator is configured on the wall of the fluid channel, and bulk acoustic waves with the propagation direction vertical to the wall are generated to the opposite wall; the region constituted by the top surface may be referred to as the bulk acoustic wave action region. The thickness of the piezoelectric layer of the inventive uhf bulk acoustic resonator ranges from about 1nm to 2 um. The frequency of the ultra-high frequency bulk acoustic wave resonator of the present invention is about 0.5-50GHz, preferably about 1-10 GHz.

In the present invention, the shape of the acoustic wave action region may be any shape. In one aspect of the invention, the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator has a width of about 50-300 μm, for example about 70-150 μm. In yet another aspect of the present invention, the bulk acoustic wave generation region area of the UHF bulk acoustic wave resonator is about 1000-50000 mu m²Preferably about 5000-²。

The bulk acoustic wave generated by the ultrahigh frequency bulk acoustic wave resonator is driven by a signal of the high frequency signal generator. The pulsed voltage signal driving the resonator may be driven with pulse width modulation, which may produce any desired waveform, such as a sine wave, square wave, sawtooth wave, or triangle wave. The pulsed voltage signal may also have amplitude or frequency modulated start/stop capability to start or cancel bulk acoustic waves.

In one of its aspects, the method of the invention is used for isolating and/or detecting a plurality of target substances in a sample. For example, in the method of the present invention, a plurality of uhf bulk acoustic resonators may be disposed in the same sample-containing container as needed, and ligands corresponding to a plurality of target substances are respectively bound to the tops of the different uhf bulk acoustic resonators, whereby a plurality of target substances can be simultaneously detected on the same sample.

The present invention also provides a kit for use in the above-described method for detecting a target substance in a sample, the kit comprising:

one or more UHF bulk acoustic wave resonators for placement on one wall of a fluid channel in which bulk acoustic waves of about 0.5-50GHz frequency are generated that propagate to the opposite wall of the fluid channel, the UHF bulk acoustic wave resonators having a ligand bound to a target species at the top.

In one aspect, the kit of the invention wherein the target substance and the ligand are each an antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer.

In one aspect, the ligand in the kit of the present invention is a peptide, glycoprotein, lipoprotein, DNA, RNA, PNA, nucleotide or polysaccharide.

In one aspect, the ligand in the kit of the present invention is biotin or streptavidin, or an antigen or antibody.

In one aspect, the kit of the present invention comprises a bulk acoustic wave generating region of the UHF bulk acoustic wave resonator having an area of about 1000-50000 μm²Preferably about 5000-²。

In one aspect, the power of the bulk acoustic wave generated by the uhf bulk acoustic wave resonator in the kit of the present invention is about 0.01 to 20mW, preferably 0.1 to 10mW, and more preferably 0.5 to 5 mW.

In one aspect, the uhf bulk acoustic wave resonator in the kit of the present invention is a film bulk acoustic wave resonator or a solid-state assembly type resonator, such as a thickness extensional vibration mode acoustic wave resonator.

The present invention also provides an apparatus for use in the above-described method for detecting a target substance in a sample. In one of the aspects of the present invention, the apparatus is used for realizing the aforementioned method for purifying a target substance from a sample of the present invention.

In one aspect of the invention, the apparatus comprises:

a container for holding a solution; one or more UHF bulk acoustic resonators disposed on the wall of the vessel, the UHF bulk acoustic resonators capable of generating bulk acoustic waves at a frequency of about 0.5-50 GHz; the top of the UHF bulk acoustic wave resonator is provided with a ligand which is combined with a target substance;

and the ultrahigh frequency bulk acoustic resonator emits a bulk acoustic wave, so that the solution in the bulk acoustic wave region generates a rotational flow, and when the target substance exists in the solution, the target substance enters the rotational flow and contacts and is combined with the ligand at the top of the ultrahigh frequency bulk acoustic resonator.

In one of its aspects, the apparatus further has a power regulating device with an output power of about 0.01-20mW, preferably 0.1-10mW, more preferably 0.5-5 mW.

In one aspect of the invention, the width of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator of the device is about 50-300 μm, for example about 70-150 μm. In yet another aspect of the present invention, the bulk acoustic wave generation region area of the UHF bulk acoustic wave resonator is about 1000-50000 mu m²Preferably about 5000-²。

In one aspect of the invention, the width of the fluid channel of the device is about 200-2000 μm, preferably about 500-1500 μm.

In one aspect, the device of the invention wherein the target substance and the ligand are each an antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer.

In one aspect, the ligand in the device of the invention is a peptide, glycoprotein, lipoprotein, DNA, RNA, PNA, nucleotide or polysaccharide.

In one aspect, the ligand in the device of the invention is biotin or streptavidin.

In one aspect, the ligand in the device of the invention is an antigen or an antibody.

In one aspect, the apparatus of the present invention is characterized in that the uhf baw resonator is a film baw resonator or a solid-state mount resonator, such as a thickness extensional vibration mode acoustic resonator.

The present invention also provides an apparatus for a method of purifying a target substance from a sample. The device is a small portable device. In one of the aspects of the present invention, the apparatus is used to realize the aforementioned method for purifying a target substance from a sample of the present invention.

In one aspect of the invention, the apparatus comprises:

a housing;

a fluid channel having an inlet and an outlet;

one or more UHF bulk acoustic resonators disposed at the bottom of the flow channel, the UHF bulk acoustic resonators capable of generating bulk acoustic waves having a frequency of about 0.5-50GHz directed at the top of the flow channel; the top of the UHF bulk acoustic wave resonator is provided with a ligand which is combined with a target substance;

In one aspect of the invention, the outlet of the fluid channel has a closure mechanism. In yet another aspect of the invention, the closure mechanism is removable. The device can be used for sealing the outlet after the sample enters the flow channel, so that the liquid in the fluid channel is static, and then the ultrahigh frequency bulk acoustic wave resonator is started to generate vortex. In yet another aspect of the invention, the closing mechanism is a chamber communicating with the outlet but isolated from the outside. The cavity is provided with a pressing part made of deformable materials, and the cavity deforms when the pressing part is subjected to pressure and the pressure is cancelled.

In yet another aspect of the present invention, the inlet of the fluid channel may also be closed by a detachable structure, such as a cap.

In one aspect of the invention, the uhf resonator has a circuit that receives a pulsed voltage signal. In yet another aspect of the invention, the uhf resonator has circuitry to accept connections to an external hf signal generator and an interface, which is located on the device housing. In another aspect of the invention, the uhf resonator has a circuit that receives a radio frequency signal.

In one aspect of the present invention, the bulk acoustic wave power generated by the uhf resonator is about 0.01-20mW, preferably 0.1-10mW, and more preferably 0.5-5 mW.

In one of its aspects, the device is further provided with a viewing window. The observation window can be observed by human eyes or can be used for being connected with an external detection instrument and used for observing signals released by the target substances enriched and combined on the surface of the ultrahigh frequency resonator, such as luminescent signals such as fluorescence.

In one aspect, the uhf bulk acoustic wave resonator in the device of the present invention is a film bulk acoustic resonator or a solid-state mount resonator, such as an acoustic resonator with a thickness extensional vibration mode.

The invention also provides a method for detecting the target substance in the sample by using the kit, the equipment or the device. The methods, kits, devices or apparatus provided herein can be used to detect virtually any analyte in vivo or ex vivo. Although in a preferred embodiment the analyte may be a biomolecule, it is not necessarily so limited as long as specific binding partners are available or some other property of the analyte can be determined in some embodiments of the invention described herein. Suitable analytes include virtually any analyte found in biological materials or materials processed therefrom. Virtually any analyte that can preferably be suspended or dissolved in an aqueous solution can be detected using the methods of the invention. Examples of analytes of interest include 1) antibodies, such as antibodies against hepatitis (e.g., hepatitis a, b, and c), measles, mumps, and rubella; 2) drugs of abuse and their metabolic byproducts, such as cotinine, cocaine, benzoylecgonine, benzoizazapine, tetrahydrocannabinol, nicotine, ethanol; 3) therapeutic agents including theophylline, phenytoin, acetaminophen, lithium, diazepam, nortriptyline, secobarbital, phenobarbital, and the like; 4) hormones and growth factors such as testosterone, estradiol, 17-hydroxypregnanolone, progesterone, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, alpha transforming growth factor, epidermal growth factor, insulin-like growth factors I and II, growth hormone release inhibitor, and sex hormone binding globulin; and 5) other analytes, including glucose, cholesterol, caffeine, corticosteroid binding globulin, DHEA binding glycoprotein, and the like.

As used in this disclosure, the term "ligand" or "analyte" or "label" refers to any molecule that is assayed. By its interaction with an anti-ligand, which binds specifically or non-specifically to the ligand, or by the characteristic dielectric properties of the ligand. A ligand is generally defined as any molecule for which there is another molecule that binds specifically or non-specifically to the ligand (i.e., an anti-ligand) due to recognition of certain portions of the ligand. An anti-ligand, for example, may be an antibody and a ligand molecule, for example an antigen, that specifically binds to the antibody. In the case where the antigen is bound to a surface and the antibody is the molecule to be detected, for the purpose of the present invention, the antibody becomes a ligand and the antigen is an anti-ligand. Ligands may also include cells, cell membranes, organelles, and synthetic analogs thereof.

Suitable ligands for use in the practice of the present invention include, but are not limited to, antibodies (forming antibody/epitope complexes), antigens, nucleic acids (e.g., natural or synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA, etc.), lectins, sugars (e.g., forming lectin/sugar complexes), glycoproteins, receptors and their associated ligands (e.g., growth factors and their associated receptors, cytokines and their associated receptors, signaling receptors, etc.), small molecules, such as drug candidates (either from natural products or synthetic analogs developed and stored in combinatorial laboratories), metabolites, drugs of abuse, and their metabolic byproducts; cofactors, such as vitamins and other naturally occurring and synthetic compounds, oxygen and other gases found in physiological fluids, cellular components, cell membranes and related structures, other natural products found in plant and animal sources, other partially or fully synthetic products, and the like.

As used herein, the term "anti-ligand" refers to a molecule that specifically or non-specifically binds to another molecule (i.e., a ligand). Anti-ligands are determined by their interaction with the ligand to which they specifically bind or by their own characteristic dielectric properties. As used herein, an antiligand is typically immobilized on a surface, either alone or as a member of a binding partner immobilized on a surface. In some embodiments, the anti-ligand may consist of a molecule on the surface of a signaling circuit or conductor. Alternatively, once the anti-ligand is bound to the ligand, the resulting anti-ligand/ligand complex may be considered an anti-ligand for the following binding purposes.

As used herein, the term "specific binding," when referring to a protein or polypeptide, nucleic acid, or receptor or other binding partner described herein, refers to a binding reaction that is determinative of the relevant ligand of interest in a heterogeneous population of proteins and/or other biological substances. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody binds to its particular "target" (e.g., hormone specifically binds to its receptor) and does not bind in significant amounts to other proteins present in the sample or to other proteins to which the ligand or antibody may be exposed in an organ or sample from an organism. Similarly, nucleic acids may hybridize to each other under preselected conditions.

As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interaction that exists between an immunoglobulin molecule and an antigen to which the immunoglobulin specifically binds. As used herein, a biological sample is a sample of biological tissue or fluid for a healthy and/or pathological state for the analyte test of interest. Such samples include, but are not limited to, sputum, amniotic fluid, blood cells (e.g., leukocytes), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therein. Biological samples may also include tissue sections, such as frozen sections for histological purposes. Although the sample is typically taken from a human patient, the test can be used to determine an analyte of interest in a sample from any mammal, such as dogs, cats, sheep, cattle and pigs. The sample may be pretreated as required by dilution in a suitable buffer solution or concentration if required. A variety of standard aqueous buffer solutions may be used, with one of a variety of buffers, such as phosphate, Tr is, or the like, preferably at physiological pH.

As used herein, the term "binding" refers to the interaction or correlation between the smallest two molecular structures, e.g., a ligand and an antiligand. This interaction can occur when two molecular structures are in direct or indirect physical contact or when two structures are physically separated but electromagnetically coupled to each other. Examples of binding interactions of interest in pharmaceutical concepts include, but are not limited to, ligand/receptor, antigen/antibody, enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid mismatches, complementary nucleic acids, and nucleic acids/proteins. Alternatively, the term "binding" may refer to a single molecule or molecular structure, such as a ligand or anti-ligand/ligand complex, described herein that binds to a signaling circuit. In this case, the signal circuit is of the second molecular structure.

As used herein, the term "ligand/anti-ligand complex" refers to a ligand that binds to an anti-ligand. The binding may be specific or non-specific, the bonds being typically covalent, hydrogen, immunological, van der waals or other types of binding.

As used herein, the term "coupled" refers to the transfer of energy between two structures either by direct or indirect physical connection or by any form of signal connection, such as electrostatic or electromagnetic coupling.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 shows a schematic diagram of an exemplary embodiment of the method of the present invention. It shows a schematic of a target substance (streptavidin) -ligand (biotin) specifically bound on top of the uhf bulk acoustic wave resonator.

Figure 2 illustrates that the method and apparatus of the present invention can accumulate micro-scale polystyrene particles and nano-scale molecules by bulk acoustic wave induced vortex.

Figure 3 shows that the method and apparatus of the present invention can deliver small biological molecules (streptavidin) in solution to the top of the uhf baw resonator through baw induced vortexing to bind and detect the ligand (biotin).

Fig. 4 shows a schematic view of an exemplary embodiment of the portable device for detecting a target substance in a sample of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Preparing a fluid channel and an ultrahigh frequency bulk acoustic wave resonator:

the fluid channel made of Polydimethylsiloxane (PDMS) was prepared by soft lithography.

And (3) carrying out chemical vapor deposition, metal sputtering and photoetching on the silicon-based wafer to finish the manufacture of the ultrahigh frequency bulk acoustic wave resonator chip. The specific method comprises the following steps:

1. the surface of the silicon wafer is thoroughly cleaned by using the piranha solution with the volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:1, and organic matters and inorganic matters on the silicon wafer can be effectively removed by the method.

2. On the cleaned silicon chip, a layer of aluminum nitride film is formed by a surface sputtering method, and then a layer of silicon dioxide film is deposited by an ion enhanced chemical vapor deposition method. Then, using the same method, aluminum nitride film and silicon dioxide film are deposited alternately to form the Bragg acoustic reflection structure with aluminum nitride and silicon dioxide alternately overlapped.

3. And sputtering a 600nm molybdenum film as a bottom electrode on the Bragg reflection layer structure. And then, photoetching the molybdenum electrode film by adopting a standard photoetching technology including glue spreading, exposure, development and the like, and then etching to form a bottom electrode with a target pattern.

4. And sputtering an aluminum nitride film on the molybdenum electrode as a piezoelectric layer. The aluminum nitride film is patterned using dry etching. In the present exemplary embodiment, a pentagon shape is employed.

5. The negative photoresist is used for transferring the pattern on the mask plate, and then a layer of titanium-tungsten alloy with the thickness of 50nm is sputtered out and used as an adhesion layer to increase the adhesion of the gold electrode. Then, a layer of 300nm thick gold film is grown on the upper electrode by using an evaporation method. And finally, removing the gold film around the target pattern by using acetone to form a gold electrode with the target pattern.

And finally, adhering and integrating the ultrahigh frequency bulk acoustic wave resonator device and the PDMS micro-channel chip. The UHF bulk acoustic wave resonator device is arranged in the middle of the channel.

The resonator device is connected with a network analyzer through a standard SMA interface, and a resonance peak is found through testing a frequency spectrum, so that the frequency of a bulk acoustic wave emitted by the ultrahigh frequency resonator device in a micro channel is measured to be 2.5GHz in the exemplary embodiment.

Example 2

The method and apparatus of the present invention employ a uhf resonator that induces eddy currents in the solution by emitting bulk acoustic waves. The particles in the solution are subjected to both acoustic radiation forces and drag forces. The micron-sized particles are large in particle size and mainly influenced by the acoustic radiation force, and can move from outside to inside along the vortex, when the micron-sized particles move to the edge of the resonator, the central liquid continues to move from bottom to top at the moment, the particles with large particle size are pushed to an area deviated from the inner side of the vortex track due to the horizontal component of the large acoustic radiation force, and after the particles are away from the edge of the device for a certain distance, the acoustic radiation force is reduced due to the fact that the distance is increased, the particles can move to the edge of the device again due to the fluid dragging force of the micro-vortex from top to bottom, and the process is repeated. When the particle size is small, the influence of dragging force is small, the aggregation is difficult, and the time required for aggregation is longer.

Two types of particles were tested in this experiment: one type is micron-sized polystyrene microspheres, the sizes of the polystyrene microspheres are 5um and 9um respectively, and the aggregation effect of particles with different particle sizes is researched by observing the time required for the particles to be aggregated at the edges of the resonator. The particle size of the micron-scale particles is larger, and the movement of the particles can be observed under normal light. The other is nano-scale particles, which are difficult to observe under normal light due to the small particle size of the nanoparticles, and Polylysine (PLL) with a fluorescent label is used for experiments: PLL-FITC, molecular weight about 250000, studied the effect of nanoparticle aggregation by observing the increase in fluorescence intensity at the resonator edges and time.

Results As shown in FIG. 2, FIGS. 2 (a-d) are the effect of aggregation of 9um polystyrene particles over time. As can be seen, after 5 minutes, the edges of the resonator have accumulated a large number of microspheres. Fig. 2 (e-h) shows the aggregation of 5um polystyrene microspheres, and at 5 minutes, some aggregation at the edge of the device can be seen, and comparing the d-plots clearly shows that 9um particles aggregate more quickly and more. FIG. 2(i-l) is the result of PLL-FITC aggregation. It can be seen that after 5 minutes, the fluorescence intensity at the edge of the device only slightly changes, and the fluorescence intensity slightly increases with the increase of time, and until after 1 hour, a more obvious fluorescence enhancement effect can be seen.

The results demonstrate that even smaller molecules, with dimensions less than 100nm, can enter the vortices generated in solution by the hypersonic bulk acoustic wave.

Example 3

The surface of the ultrahigh frequency acoustic wave resonator is modified by PLL-biotin to generate the surface of the ultrahigh frequency acoustic wave resonator with biotin molecules, and streptavidin marked by Fluorescence (FITC) is added into the solution. For the control group, no power was applied, and the target substance (streptavidin) -ligand (biotin) was bound and reacted by free diffusion. The reaction of target substance (streptavidin) -ligand (biotin) after generating vortex on the surface of the UHF acoustic wave resonator was observed with 1mW power applied to the experimental group.

The specific experimental procedure is as follows:

(1) a1 mg/ml PLL-PEG-biotin solution and 200nM fluorescein isothiocyanate labeled streptavidin (FITC-SAv) were prepared.

(2) And (3) placing the ultra-high frequency acoustic resonator into piranha washing liquor (solution with volume ratio of concentrated sulfuric acid to hydrogen peroxide being three to one) for washing for 15 s.

(3) The washed chip was incubated in a PLL-PEG-biotin solution for 30 minutes at room temperature.

(4) After adding 50ml of FITC-SAv solution and applying 1mW power (experimental group) or no power (control group), the fluorescence intensity was recorded by a video camera (Olympus DP73, Japan) with a fluorescence microscope (Olympus BX53, Japan).

During the experiment, the detection results are photographed after 25 minutes, 30 minutes and 40 minutes, respectively. Fig. 3 (a-d) shows the signal for the surface of the resonator without the resonator emitting bulk acoustic waves and creating eddy currents in the solution. It can be seen that after 40 minutes there is no increase in fluorescence on the resonator, i.e. the biotin reacts less with streptavidin and the lowest threshold of detection is not reached. Fig. 3 (e-h) is an observation of the resonator emitting bulk acoustic waves and creating vortices in the solution. It is evident that the intensity of the fluorescence increases with time, forming a pentagonal aperture at the edge of the resonator.

Experiments prove that in the method and the device, the small molecules in the solution can be delivered to the surface of the ultrahigh frequency resonator through the acoustic vortex and combined with the ligand of the target molecules coated on the surface, so that the purpose of capturing and detecting the small molecules in the solution is achieved.

The inventors believe that one of the principles of this unexpected discovery is that a small molecule of a target substance that can specifically bind to a ligand in a solution is bound to the ligand at an interface surface with the ligand, and if it contacts and binds to the ligand by free diffusion of the small molecule of the target substance in a liquid alone, it takes a long time to wait; by the method and the device, the biological small molecules in the solution can be rapidly delivered to the surface of the ultrasound resonator by utilizing the eddy current formed by the ultrasound bulk acoustic wave in the solution (one end of the trace of the eddy current is close to or in contact with the surface of the ultrasound resonator), so that the contact chance among the molecules is greatly enhanced, and the process of combining and detecting is greatly accelerated. Moreover, because the target substance micromolecules to be detected in the solution are continuously conveyed to the vortex range, other micromolecules continue to enter after the micromolecules entering the vortex range are combined on the ligand; the small molecules of the target substance to be detected in the whole container have the opportunity to enter the vortex and combine with the ligand, which is equivalent to enriching and supplementing all the small molecules of the target substance to be detected in the solution.

Therefore, the method and the device of the invention not only can accelerate the detection time of the biological micromolecules, but also can greatly improve the detection sensitivity.

Example 4

The method of the invention is based on a device with an ultra-high frequency resonator. The ultrahigh frequency resonator is processed by adopting MEMS compatible with a semiconductor process, so that the miniaturization and the integration are facilitated. The equipment provided by the aspect is small in size and simple to operate.

The invention provides a small portable device for detecting a target substance in a sample, which has a schematic structural diagram as shown in FIG. 4. The device has a housing 101 and a fluid chamber 102 within the housing having an inlet 108 and an outlet 103. Above the outlet 103 is placed a chamber 104 which is in communication with the outlet 103 but is isolated from the environment. The liquid sample 107 may be directly overlaid on the inlet 109, such as by a dropper or applicator. Applying a certain pressure on the cavity 104 by fingers, so that the cavity is subjected to volume reduction deformation; the sample 107 is then placed over the inlet 108 and the finger is released slightly, the volume of the chamber returns to its original size, the sample 107 is drawn into the microchannel chamber 102 by the external atmospheric pressure, and the uhf baw resonator 105 disposed at the bottom of the chamber is activated to generate eddy currents 106 that deliver the target substance in the sample to the surface of the uhf baw resonator that has a ligand that specifically binds to the target substance. The signal released from the target substance captured by the ligand, for example, a luminescence signal such as fluorescence, can be directly observed by the human eye through an observation window (not shown), and can also be used in connection with an external detection instrument.

The uhf resonator in the above-described device has a circuit that receives a pulsed voltage signal. For example, the uhf resonator has a circuit for receiving a connection to an external hf signal generator and an interface, which is provided on the device housing.

For another example, the uhf resonator has a circuit that accepts a radio frequency signal. The emission of bulk acoustic waves is excited or stopped by a wireless signal.

The UHF bulk acoustic wave resonator can generate high-frequency (about 0.5-50GHz) vibration to induce bulk acoustic waves with corresponding frequencies in a solution. In one aspect of the present invention, the ultra high frequency bulk acoustic resonator is a Film Bulk Acoustic Resonator (FBAR) or a solid state fabricated resonator (SMR), preferably a solid state fabricated resonator. In yet another aspect of the present invention, the uhf bulk acoustic resonator is an acoustic resonator of a thickness extensional vibration mode, and the thin film layer of piezoelectric material is grown in a vertical direction, and the vibration is excited by coupling a vertical electric field with a d33 piezoelectric coefficient. The ultrahigh frequency bulk acoustic wave resonator adopted by the invention can generate localized acoustic current at the interface of the device and liquid without the help of a coupling medium or a structure. The acoustic wave resonator comprises an acoustic wave reflecting layer, a bottom electrode layer, a piezoelectric layer and a top electrode layer which are sequentially arranged from bottom to top. The overlapped area of the bottom electrode layer, the piezoelectric layer, the top electrode layer and the acoustic wave reflecting layer forms a bulk acoustic wave generating area. The top surface of the ultrahigh frequency bulk acoustic wave resonator is configured on the wall of the fluid channel, and bulk acoustic waves with the propagation direction vertical to the wall are generated to the opposite wall; the region constituted by the top surface may be referred to as the bulk acoustic wave action region. The thickness of the piezoelectric layer of the inventive uhf bulk acoustic resonator ranges from about 1nm to 2 um. The frequency of the ultra-high frequency bulk acoustic wave resonator of the present invention is about 0.5-50GHz, preferably about 1-10 GHz.

The bulk acoustic wave power generated by the UHF resonator is about 0.01-20mW, preferably 0.1-10mW, and more preferably 0.5-5 mW. The width of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator is about 50-300 μm, for example about 70-150 μm.

Although exemplary embodiments of the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. For other examples, one of ordinary skill in the art will readily appreciate that the order of the process steps may be varied while maintaining the scope of the present invention.

Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

The following papers are hereby incorporated by reference in their entirety.

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Claims

1. A method of detecting a target substance in a sample, the method comprising:

processing a sample to be tested in an apparatus comprising; a container for holding a solution; one or more UHF bulk acoustic resonators disposed on one wall of the container, the UHF bulk acoustic resonators capable of generating bulk acoustic waves having a frequency of about 0.5-50 GHz; the top of the UHF bulk acoustic wave resonator is provided with a ligand which is combined with a target substance;

2. The method of claim 1, comprising the step of detecting an identifiable label carried by the target substance, such as fluorescence or other forms of luminescence (e.g., chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallography, thermoluminescence, sonoluminescence, phosphorescence, photoluminescence, etc.), enzymatic reactions, radioactivity, etc.

3. The method of claim 1, wherein the target substance and the ligand are each an antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer,

for example, wherein the target substance is biotin or streptavidin, or an antibody or antigen.

4. The method as claimed in claim 1, wherein the bulk acoustic wave generation region area of the UHF bulk acoustic wave resonator is about 1000-50000 μm²Preferably about 5000-²。

5. The method of claim 1, wherein the bulk acoustic wave generated by the uhf bulk acoustic wave resonator has a power of about 0.01-20mW, preferably 0.1-10mW, more preferably 0.5-5 mW.

6. A kit for use in the method for detecting a target substance in a sample according to any one of claims 1 to 5, the kit comprising:

one or more UHF bulk acoustic wave resonators for placement on one wall of a fluid channel in which bulk acoustic waves of about 0.5-50GHz frequency are generated that pass to the opposite wall of the fluid channel, the UHF bulk acoustic wave resonators having a ligand bound to a target species on top.

7. The kit of claim 6, wherein the target substance and the ligand are each an antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, or repressor/inducer,

for example, wherein the ligand is biotin or streptavidin, or is an antigen or antibody.

8. The apparatus for use in the method for detecting a target substance in a sample according to any one of claims 1 to 5, comprising:

9. The device of claim 8 having a power regulating means for regulating the power of the bulk acoustic wave generated by the uhf resonator, preferably wherein the output power of the power regulating means is about 0.01-20mW, preferably 0.1-10mW, more preferably 0.5-5 mW.

10. A device for detecting a target substance in a sample, the device comprising:

a housing;

a fluid channel having an inlet and an outlet;