Integrated MEMS sensor with bridged conformation shift receptors

Abstract

An integrated NEMS cantilever sensor with a precisely controlled cantilever gap has a receptor mounted to bridge the gap. When the receptor interacts with the target analyte, it changes its conformation and induces a change in the gap. A detector within the sensor generates a signal corresponding to the change in gap dimension, confirming the interaction with the analyte.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 488,865, filed Mar. 7, 2023, which is hereby incorporated by reference in its entirety.

[0002] This application generally relates to devices comprising microelectromechanical system (MEMS) sensors, and more particularly to devices and methods for the detection, identification, and quantification of target analytes.

Background Art

[0003] State-of-the-art analyte diagnostic techniques utilize fundamental chemistry and analytical instruments (such as spectrophotometry, gas or liquid chromatography). In the context of this discussion, an analyte is defined as any inorganic or organic compound or substance for which information is sought, which may include identity, purity, quantity, and / or concentration. For example, currently used pathogen sensing techniques utilize enzyme-based immunoassays, polymerase chain reaction (PCR), fluorescence signaling, or quartz microbalances and silicon microcantilevers to determine the presence and / or mass of a target pathogen. Unfortunately, state-of-the-art techniques often lack the speed, accuracy, and sensitivity to provide end-users with sufficient information-based decision support. The presence of background substances typically impairs the accuracy of these techniques and thus requires extensive sample preparation prior to diagnosis. Additionally, these techniques are very cumbersome and complex to operate, making false positive and false negative results likely.

[0004] Meeting the needs of the modern analyte diagnostic market means developing technologies that are sensitive enough to accurately detect a relatively small number of molecules as quickly as possible and with ambivalence to environmental background substances. New materials, nanotechnology, and miniature biomechanical devices have enabled great progress towards this goal.

[0005] Microelectromechanical systems (MEMS) are promising technologies that, by combining silicon-based microelectronics with micromachining techniques, have the potential to revolutionize both industrial and consumer products. MEMS have several distinct advantages as a manufacturing technology, such as the interdisciplinary nature of MEMS technology and its micromachining techniques, and the fact that its diverse applications have led to an unprecedented range of devices and synergies across previously unrelated fields (such as biology and microelectronics). In addition, MEMS batch manufacturing techniques enable the production of components and devices with improved performance and reliability in configurations with reduced physical size, volume, weight, and cost. Furthermore, MEMS provide the basis for manufacturing products that could not otherwise be made. These factors make MEMS potentially a much superior detection technology.

[0006] Currently, most MEMS applications are found in systems across automotive, electro-optical, communication, and defense applications. Due to their unique mechanical properties and small size, MEMS sensors are ideal for use, among other things, as accelerometers for airbag sensors, inkjet printer heads, computer disk drive read / write heads, projection display chips, blood pressure sensors, optical switches, and microvalves.

[0007] Improvements in MEMS processing technology have significantly facilitated the development of biosensors for medical applications. Such improvements include bulk surface micromachining, which selectively removes portions of silicon or adds additional structural layers to form mechanical and electromechanical components. MEMS-based biosensors can utilize not only the electrical properties of silicon, but also either the mechanical properties of silicon or both its electrical and mechanical properties.

[0008] Cantilevers are one of several fundamental components of MEMS with dimensions in the micro or nano range. Cantilevers can be fabricated from silicon (Si), silicon nitride (Si3N4), or polymers using bulk micromachining, surface micromachining, or a combination of both. In each micromachining process, the solid structure is freed from the substrate to create a self-supporting beam fixed at one end. Cantilevers can be fabricated as a single unit or as an array of multiple units and can be designed to have varying degrees of "rigidity" or flexibility to meet the requirements of a particular application. These processes also allow for the fabrication and integration of electronic circuits and other MEMS components necessary to interface with the cantilever. Due to their flexibility and versatility, these components have become promising components used in a variety of environmental, biomedical, and consumer product applications. In prior art biosensing applications, MEMS cantilevers are often integrated into test chambers to isolate receptor-analyte reactions in a minimum volume.

[0009] In conventional MEMS sensor cantilevers, bending in both static and dynamic modes is achieved by varying the mass and / or Young's modulus. The dynamic mode utilizes vibration as the driving force, in contrast to gravity in the static mode. However, these conventional MEMS cantilevers have significant limitations.

[0010] Cantilever sensors can operate in static and / or dynamic modes. In static mode, molecular interactions on the cantilever surface are converted into cantilever bending as a result of changes in surface stress. In static mode, displacement or deflection within the cantilever can be measured using various techniques, such as diffracted or reflected light, or piezoresistive materials. In dynamic mode, changes in mass or Young's modulus can be measured by the resonant frequency of the vibrating cantilever. Young's modulus can induce negative or positive frequency shifts depending on the thickness of the analyte deposit layer. Dynamic mode is more sensitive to changes in cantilever properties than static mode. For biocantilever devices known in the art, it should be noted that a sufficient mass must be deposited on the device to affect the detectable response. Furthermore, nonspecific deposition from background material is a well-known challenge for such biocantilever devices, requiring masking of the cantilever surface or preparation or purification of the sample to achieve sufficient specificity.

[0011] The dynamic mode can be explained by the following formula.

[0012] The equation for the force equilibrium of a cantilever is as follows:

number

number

[0013] When we find the derivative,

number

[0014] The resonant frequencies of the cantilever before any mass is added are as follows:

number

number

[0015] By taking the logarithm and then differentiating, equation (3) is transformed to obtain equation 5:

number

[0016] If the spring constant does not change during mass deposition, the change in frequency is a function of the change in deposited mass, so Equation 5 can be simplified as follows:

number

[0017] Substituting into the above equation, equation 6 becomes as follows:

number

number

[0018] The mass associated with the cantilever before adding mass is the volume V b multiplied by the density ρ and the geometric coefficient α1. For a straight beam clamped at one end, α1 = 0.24 V b = LWH, and m b = α1ρWLH

[0019] Using these relationships, Equation (7) becomes as follows [Number]

[0020] The change in the resonance frequency is directly proportional to the product of the change in mass and the group of constants that define the characteristics of the cantilever

[0021] When the change in the mass load on the cantilever can be ignored compared to the spring constant, Equation (5) is simplified as follows [Number] where [Number] is

[0022] Determining the change in the spring constant of the cantilever as a function of the height of the analyte deposited or adsorbed on the surface is achieved by taking the derivative of k with respect to H in Equation 9, which provides an approximation for small ΔH. The additional analyte basically changes the Young's modulus or the cantilever stiffness [Number] where dH ~ N s A t H t and N sA is the number of analyte components on the surface of the cantilever. t This is a surface covered with the analyte components, and H t This represents the height of each individual analyte component. For typical applications reported in the literature, the cantilever constant can be varied by an order of magnitude from 5% to a maximum of 40%.

[0023] The cantilever's response to a change in mass in response to a change in Young's modulus can be calculated. The relative frequency changes due to the mass loading (negative) and Young's modulus (positive) contribute equally to the same point. This condition is met when there is no obvious resonant frequency shift due to the addition of analyte components to the cantilever surface, i.e., ω 0、b =ω 0、a ,or

number

[0024] The masses of the cantilever before and after the addition of mass are, respectively: m b =α1ρ b WLH, and m a =α1ρ b WL(H+dH c ) and here, H c This is the critical height of the analyte component layer.

[0025] From equation (9) above, the spring constant k before the addition of the additional analyte material is given by: After the addition of the analyte, the spring constant becomes as follows:

number

number

[0026] MEMS cantilevers in aqueous solutions, i.e., MEMS cantilevers not in air, present further detection challenges due to a significant damping effect on the dynamic motion of the cantilever, where γ>>0. The solution to Equation 1 is as follows:

number

number

number

[0027] Static mode detection does not induce oscillations so that acceleration or velocity terms are not introduced into the force equilibrium system. For a typical cantilever spring, this is on the order of several times 10,000 G at resonant frequencies in the MHz range. In static modes, the force on the cantilever is from the Earth's gravitational field, where the force is simply F=mg, and g is the acceleration due to gravity.

[0028] Therefore, the force equilibrium equation for a cantilever operating in static mode is ky = F ext It is simplified to =mg.

[0029] Taking the logarithm and differentiating, we obtain the following:

number

number

[0030] Similar to dynamic modes, the displacement of a static mode system is a function of both the change in mass and the change in the spring constant.

[0031] For a cantilever spring system where the spring constant does not change, equation (18) can be reorganized with respect to the displacement due to the addition of mass, i.e.,

number

[0032] Because the change in total mass due to a typical mass load on a MEMS cantilever is extremely small, the displacement or deflection of the cantilever is extremely small, on the order of femtometers (fm). The magnitude of this displacement does not fit with known conversion methods. Without acceleration and velocity terms contributing to the equation of motion, the purely static detection mode is significantly inferior to the dynamic detection mode.

[0033] The relative change in the spring constant that affects displacement when the relative mass load is small compared to the change in the spring constant is as follows:

number

[0034] Regardless of mass loading, changes in Young's modulus due to surface material modification are extremely difficult to measure. Therefore, small displacements associated with purely static mode methods are not suitable for detection schemes.

[0035] Nevertheless, the static mode can be improved by effectively reducing the apparent cantilever spring constant using an additional force. Applying an electric force through a capacitor configuration can significantly improve the response to changes in mass load, however, changing the effective spring constant is limited by the highly nonlinear behavior of the electrodynamics of the changing plate distance (the gap from the cantilever to the base).

[0036] To improve performance, the cantilever surface can be further coated or functionalized. To explore specific analytes in a sample, the cantilever surface may be coated or functionalized with a layer of specific chemical or biomolecules. Since harsh deposition and etching processes can impair any existing functionalization, cantilever surface functionalization is usually performed after the sensor has been fabricated. The functional layer can be coated on one side of the cantilever surface or on any specific localized area of ​​the cantilever surface. The quality of the functionalization directly affects the performance of the sensor signal.

[0037] Cantilever surface modification for chemical, biological, and biomedical applications is classified into two categories: physical and chemical. Physical modification, such as etching, grit blasting, and machining, results in changes to the surface topography or morphology with little to no change in chemical properties. Established chemical techniques include plasma and chemical vapor deposition, atomic layer deposition, and electrochemical deposition. Chemical treatments can result in surface oxidation / nitriding / carbonization, surface functionalization, ion implantation, single-layer coating, or coatings containing multiple layers of different compositions. The objectives of modifying a cantilever surface may include (1) generating a specific surface topography, (2) improving biocompatibility, (3) modifying the surface composition, or (4) creating layers of material with a specific chemical composition.

[0038] Many tools have been developed to address the challenges of functionalizing cantilever surfaces. Microcapillaries are relatively easy to use and suitable for functionalizing a small number of cantilevers. A micromanipulator with a translational stage precisely positions a microcapillary tube filled with chemicals to approach the cantilever sensor. The capillary tube allows a specific cantilever sensor to come into contact with the chemical solution for functionalization. Another efficient method for functionalizing cantilevers is to use chemical inkjet printing techniques, which can be used to fabricate high-density DNA and protein microarrays. Functionalization of self-assembled monolayers, polymer solutions, and DNA samples has been demonstrated to have comparable performance on cantilever sensors. Using this method, microcantilever sensors can be batch functionalized at the wafer level. Other potential modifications of cantilevers are well known; for example, the cantilever surface can be modified to form one or more porous silicon regions exhibiting different morphological, chemical, thermomechanical, and photonic properties. An example of porous silicon treatment is provided in U.S. Patent No. 7,433,811 by Gao, et al., which is incorporated herein by reference.

[0039] A further expansion of the use of MEMS as sensors is biological microelectromechanical systems: namely, bio-MEMS, which involve the use of small biological-mechanical structures that respond to environmental changes. These changes may include the introduction of organic or inorganic analytes, or thermal changes in the environment. The biological components of bio-MEMS, called "receptors," respond to environmental changes, and this response can be translated into the movement of mechanical structures.

[0040] The relevant technologies in bio-MEMS sensors include various device designs, signal generation and conversion modes, and the orientation of biological components. Generally, the upper surface of a MEMS cantilever device is bound to a receptor molecule that specifically binds to a unique target molecule. For example, an immobilized antibody receptor interacts with a specific antigen target. Thus, the receptor-target complex significantly alters the mass of the MEMS device, resulting in changes in the orientation, bending, or resonance of the MEMS. Bio-MEMS can operate in either static or dynamic modes. In static mode, the association of target molecules to the receptor-coated surface generates stress that induces bending of the cantilever. In dynamic mode, the resonance frequency of the cantilever decreases as target molecules adsorb onto the receptor-coated surface. Either approach to detection requires a considerable number of receptor-target complex molecules to induce significant changes in the position and / or resonance of the MEMS sensor. These technologies may lack specificity, exhibit very low sensitivity, and are extremely difficult to manufacture consistently on a large scale.

[0041] The means of signal conversion or cantilever deflection measurement can be interferometry or optical beam deflection from the free end of the cantilever, the latter typically found in AFM (atomic force microscope) instruments. This approach requires a laser light source and a position-sensing photodetector, which tend to require frequent calibration and a consistent sample refractive index. As a result, this approach often fails in applications outside of a controlled laboratory environment. Another common approach may involve coating the cantilever with a piezoresistive material, thereby inducing a change in conductivity and / or resistance through stress.

[0042] Other prior art envisions various molecules for crosslinking the gap between opposing electrodes. For example, Albert, et al. US8,078,408 and US9,857,366 (incorporated herein by reference), and Merriman, et al. US10,036,064 and US2019 / 0094175 teach crosslinked electrodes that complete a circuit and / or suppress resonance as a result of conductive high A / G composition DNA. In Merriman's disclosure, the association of the crosslinking molecule with molecules in its environment does not induce mechanical bending of the electrode(s). Albert's '408' and '366' patents describe crosslinking the gap between the free ends of two surfaces with a template molecule of ssDNA to form a circuit. Upon hybridization with the target molecule, the dsDNA increases the conductivity of the entire circuit. The description provided refers to an alternative approach to measuring cantilever deflection using piezoresistive resistance, but the lack of a viable level of disclosure, combined with the inventor's subsequent inability to manufacture a functional device with any degree of repeatability in the gap and behavior, led to the abandonment of this endeavor.

[0043] Among the many types of molecular interactions is nucleic acid hybridization, which occurs when a single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule is annealed and hybridizes to a complementary DNA or RNA molecule. Relevant methods and devices for detecting biological targets have applied the basic principle of hybridization between complementary nucleic acid base pairs (e.g., cytosine binding to guanine, and adenine binding to thymine or uracil). Typically, a method is known in which a specific sequence of nucleotides, bound to a radioactive, fluorescent, or chromogenic tag, is exposed to an array of nucleic acid molecular fragments covalently bound to a surface or material (such as nylon or nitrocellulose). If the nucleotide sequence of the tagged "probe" is complementary to a fragment in the array of molecules bound to the surface, hybridization occurs, and detection is determined by the presence of a radioactive, fluorescent, or chromogenic signal. Generally, the aforementioned techniques for introducing hybridization require a considerable amount of analyte, resulting in a lower detection limit.

[0044] Sensing methods and devices that utilize intrinsic or inherent molecular structures and changes in molecular structure as a result of changes in the environment surrounding molecules are well known. Examples of environmental influences on molecular structure include changes in temperature, pH, salinity, and light emission or absorption. Other examples of environmental influences include the introduction of other molecules that alter the structure of commensal molecules through various interactions (i.e., molecular exclusion, hydrophilicity, ionic bonding, etc.). As an example, neuroproteins called prions can be converted from alpha-helix to beta-sheet conformation in the presence of other beta-sheet prions. Another example is hybridization to form a double-stranded DNA helix by introducing a single-stranded DNA molecule in the presence of a complementary DNA strand. It is well known in the art that double-stranded DNA molecules may exhibit inherent curvature that performs important biological functions such as nucleosome positioning and gene expression. Various theoretical models have been proposed to explain the relationship between DNA nucleotide sequences and the bending of the helical axis or inherent DNA curvature. Common to these models is the number, position, and length of adenine nucleotide tracts called A-tracts. The hydrogen bonds between guanine and cytosine residues are perpendicular to the helical axis. Conversely, the bonds between adenine and thymine residues have a significant angle of 2°–4° per base pair. Therefore, A-tracts positioned on the same side of the DNA helix can induce the inherent curvature of the molecular axis, significantly reducing the end-to-end distance of the double-stranded nucleotide sequence. [Overview of the project]

[0045] The devices and methods disclosed herein are intended for the detection of substantially any analyte, regardless of the type of sample or background material in the sample. In some embodiments, the device comprises one or more integrated sensors, the sensors configured to form a crosslinked cantilever shape. The integrated sensor comprises a first surface, a second surface, and a receptor, one of which is flexible. The receptor comprises one or more molecular structures that change conformation upon interaction, association, or dissociation with one or more analytes. A first contact point on the receptor is attached to a first surface contact point on the first surface, and a second contact point on the receptor is attached to a second surface contact point on the second surface. In some embodiments, the receptor is configured to be physically associated with the analyte when the receptor is associated with the analyte. In some embodiments, the receptor is configured to be physically dissociated from the analyte when the receptor is not associated with the analyte. The detector generates an output signal when the receptor interacts, associates, or dissociates with the analyte.

[0046] Sample preparation reagents (SPRs) and methods for using SPRs to detect analytes in samples are also provided. SPRs contain pH buffering components, enzyme activity inhibitors, denaturants, low ionic strength detergents, and oxidizing agents. The buffering components maintain pH to facilitate the dissociation of the analyte from its ligand, native complement, or intrinsic secondary structure. Low ionic strength detergents disrupt host viruses or cell wall structures, releasing the analyte from the cell. Oxidizing agents induce controlled oxidative fragmentation of the analyte.

[0047] In one embodiment, a sensor assembly for detecting a target analyte comprises: a cantilever formed from a silicon material having a deflection sensing element disposed therein, the cantilever being elongated and having a proximal end and a distal end; a support base having a silicon pedestal disposed on a support surface; a first conductive metallization disposed on the lower surface of the cantilever and in electrical contact with the deflection sensing element; a second conductive metallization disposed on the upper surface of the silicon pedestal, the second conductive metallization being configured to transmit electrical signals between the deflection sensing element and a contact for electrical communication with an external electrical device; and the first and second conductive metallization A eutectic joint formed between an electrochemical metallization and a pedestal, wherein the eutectic joint is configured to fix the proximal end of a cantilever onto the pedestal, and the pedestal has a pedestal thickness for supporting the lower surface of the distal end of the cantilever with a fixed gap from the support surface; and at least one bridging receptor having a first end and a second end, wherein the first end is configured for attachment to the lower surface of the distal end of the cantilever, and the second end is configured for attachment to the support surface, and the bridging receptor is configured to change conformation upon interaction with a target analyte, thereby inducing deflection of the cantilever.

[0048] In some embodiments, the deflection detection element comprises a plurality of piezoresistive elements formed within the silicon material of the cantilever or coated on the surface of the cantilever. The plurality of piezoresistive elements may be configured to define a Wheatstone bridge. The eutectic junction may be a gold-silicon junction. In some embodiments, the fixed gap is in the range of 1 to 1,000 nm. Metallized contact regions may be formed on the underside and support surfaces of the distal end of the cantilever for mounting the end of at least one bridging receptor. The metallized contact regions may contain gold, and at least one bridging receptor is thiol-modified to facilitate mounting to the metallized contact regions.

[0049] At least one crosslinking receptor may be an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repeat of any one of these molecules, a complex of any one of these molecules, a hybridization of any one of these molecules, or any combination thereof. In some embodiments, at least one crosslinking receptor may be a nucleic acid, a nucleic acid analog, double-stranded DNA (dsDNA), a dsDNA analog, single-stranded DNA (ssDNA), an ssDNA analog, or a peptide nucleic acid. At least one crosslinking receptor may be a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. At least one crosslinking receptor may be 50 to 1,000 nm in length and may undergo a length reduction of about 15% to about 80% upon interaction with the target analyte.

[0050] In some embodiments, the analytes detected by the sensor assembly may be inorganic molecules, organic molecules, polymers, polymer analogs, carbon nanotubes, carbohydrates, carbohydrate analogs, nucleic acids, nucleic acid analogs, proteins, protein analogs, antibodies, antibody analogs, repeats of any one of these molecules, complexes of any one of these molecules, hybridizations of any one of these molecules, or any combination thereof. The analytes may also be complementary nucleic acids, complementary nucleic acid analogs, complementary dsDNA, complementary dsDNA analogs, complementary ssDNA, complementary ssDNA analogs, complementary RNA, or complementary RNA analogs. The analytes may be ssDNA or RNA.

[0051] In another embodiment, an analyte detection system comprising: a test well or chamber configured to accept a sensor assembly as described above; and a conductive connector configured for electrical communication between the sensor assembly and an instrument for generating an external indicator showing the detected deflection of the cantilever. The test well or chamber may be configured to hold one or more samples from among tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal specimens, nasopharyngeal specimens, exhaled breath specimens, urine, feces, tissue, blood, plasma, serum, cell cultures, body fluids, tissue biopsies, apocrine or eccrine fluids, forensic specimens, aerosols, soil, water specimens, food, components, raw materials, in-process specimens, by-products, products, or quality control specimens. The specimens may include viruses, bacteria, phages, yeast, mycoplasma, fungi, human cells, animal cells, plant cells, insect cells, or any combination thereof. The specimens may be mucus specimens, nasal specimens, exhaled breath specimens, or nasopharyngeal specimens. The sample may contain a virus, which may be one or more of the following: coronavirus, influenza virus, respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or Zika virus.

[0052] The scheme of this invention utilizes the change in the length of the attached receptor from end to end. The change in the length of the receptor corresponds to the magnitude of the change in the deflection / displacement distance of the MEMS cantilever. As long as the curvature strength of the receptor exceeds the force constant of the cantilever, this approach is independent of the physical properties of the MEMS cantilever. More specifically, length, thickness, width, processed material, surface properties (Young's modulus), and mounting point are not applicable to this approach. The advantage of this approach is that the processing process, processed material, and physical properties of the MEMS cantilever are not important for the successful implementation of analyte detection and identification. It should be noted that the mounting point and / or the length of the cantilever affect the sensitivity of detection because these parameters change the magnitude of the deflection / displacement of the MEMS cantilever. The degree of change in sensitivity can be calculated from the physical properties of the trait-introducing material, i.e., the piezoresistive material. The shorter the cantilever and the closer the receptor mounting is to the clamping point, the higher the sensitivity. However, since the preferred embodiment mimics a binary system with respect to deflection and the signal is experimentally measurable in this application, sensitivity is not a parameter that requires improvement. Furthermore, because it depends on the relative change of the transformed value rather than the absolute value, the need to know the absolute initial deflection / displacement position of the MEMS cantilever and the specific value of the piezoresistive element on the MEMS cantilever is reduced. Each MEMS cantilever with a receptor can potentially have a variety of initial unreacted starting values, which are known prior to the introduction and application of the analyte. [Brief explanation of the drawing]

[0053] [Figure 1A-B] Embodiments of a bridged conformation shift receptor for static (Figures 1A-1B) and dynamic (Figures 1C-1D) modes are schematically shown. [Figure 1C-D] Embodiments of a bridged conformation shift receptor for static (Figures 1A-1B) and dynamic (Figures 1C-1D) modes are schematically shown. [Figure 2]This shows an exemplary sensor device configuration with variations in the number and / or relative orientation of bridged conformation shift receptors and cantilevers. [Figure 3] A schematic example of a cantilever sensor according to an embodiment of the scheme of the present invention is provided, where panel A shows the state before exposure, and panels B to F show the conformational changes of the cantilever caused by the tensile, compressive, rotational, torsional, and combined responses of the BCSR, respectively. [Figure 4] The graph shows peaks corresponding to potential receptor candidates, along with a calculated plot of relative curvature along the organism's genome. [Figure 5A] From left to right, the distal end of the cantilever sensor assembly is schematically shown before exposure to the analyte, after detection, and after reset. [Figure 5B] This figure shows the device's response to analyte detection and reset. [Figure 6A] This is a top view of an exemplary integrated sensor assembly according to one embodiment of the device of the present invention. [Figure 6B] This is a schematic diagram showing an exemplary Wheatstone bridge used in an embodiment of the sensor. [Figure 6C] A schematic process flow for manufacturing a cantilever arm according to one embodiment of the scheme of the present invention is shown. [Figure 6D] This diagram illustrates a process flow for fabricating a support base and assembling a cantilever and support base according to one embodiment of the scheme of the present invention. [Figure 6E] A diagram of the integrated sensor of the present invention according to one embodiment is provided. [Figure 7] A diagram is provided illustrating an exemplary detection system for use in detecting a target analyte according to one embodiment of the system of the present invention. [Figure 8A] This is a side perspective view of an optional flow cell assembly for use in one embodiment of the system of the present invention. [Figure 8B]This is a top perspective view of an optional flow cell assembly for use in one embodiment of the system of the present invention. [Figure 8C] This document illustrates an exemplary approach for exposing the integrated sensor of the present invention to a target analyte. [Figure 8D] This is a diagram of an alternative embodiment for exposing the sensor to a target. [Figure 9] This is a flowchart of the sequencing process for creating cantilever-BCSR combinations and using those combinations for pathogen detection. [Figure 10] This bar graph shows the deflection of the BCSR when the target analyte is detected. [Figure 11] This bar graph shows the consistent manufacturability of BCSR sensors. [Figure 12] This table shows the correlation between receptor-specific DNA curvature and the magnitude of cantilever deflection. [Figure 13] This table lists the details of target receptors used in the diagnostic applications of the system and method of the present invention. [Figure 14A] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14B] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14C] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14D] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14E]This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14F] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14G] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14H] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Figure 14I] This report provides bar graphs of test results and cantilever deflection data for nine different targets of varying forms against background materials. It also includes calculated structural and predicted system response versus actual system response. [Modes for carrying out the invention]

[0054] Abbreviations and definitions To facilitate understanding of the present invention, several terms and abbreviations used herein are defined as follows:

[0055] As used herein, “MEMS” refers to a microelectromechanical system that incorporates a microscale integrated device or system combining mechanical and electrical components. MEMS are generally manufactured using conventional integrated circuit (IC) processing techniques, such as photolithography, electron beam, and / or other patterning methods, and their size can range from the order of 1 μm to 1000 μm.

[0056] As used herein, "NEMS" refers to nanoelectromechanical systems, i.e., MEMS on a reduced scale, which may range from 1 nm to 1 μm. MEMS consist of mechanical nanostructures, nanosensors, nanoactuators, and microelectronics.

[0057] As used herein, "cantilever" is a structural element (beam) that extends horizontally and is supported at one end with respect to a reference plane or base. MEMS devices are generally manufactured from silicon due to its favorable material properties, but many other materials have been used, including but not limited to silicon nitride (Si3N4) and other ceramics, sapphire, quartz, silicon carbide, aluminum nitride, metals, polymers, or composites thereof.

[0058] As used herein, BCSR refers to a crosslinked conformational shift receptor which can be mounted to crosslink gaps between different surfaces of MEMS and / or NEMS structures (e.g., between the cantilever surface and a reference plane). BCSR contact points are regions on the cantilever or base. BCSR contact points can be fabricated from a variety of materials, including gold, silicon, silicon dioxide, glass, quartz, polymers, platinum, titanium, tin, aluminum, nickel, copper, and iron. Surface contact points can be functionalized with alcohol groups, alkene groups, alkyne groups, amine groups, carboxylic acid groups, aldehyde groups, ketone groups, ester groups, and ether groups. Modification of surface contact points may differ from modification of the rest of the cantilever surface for chemical, biological, and biomedical applications.

[0059] As used herein, a “receptor” (which may be interchangeably represented by a capital letter or all lowercase letters) is a substance that changes its conformation in response to a change in its environment. A receptor comprises one or more molecular structures that function as a binding site for one or more analytes. When one or more analytes bind, the receptor changes to a different conformation. A non-exclusive list of receptors includes inorganic molecules, organic molecules, polymers, polymer analogs, carbohydrates, carbohydrate analogs, nucleic acids, nucleic acid analogs, proteins, protein analogs, antibodies, antibody analogs, repeats of any one of those molecules, complexes of any one of those molecules, hybridizations of any one of those molecules, or any combination thereof.

[0060] As used herein, “sensor” is a device that detects or measures information from the surrounding environment and provides an output signal in accordance with the detected or measured parameters. A non-exclusive list of information to be detected or measured includes mechanical (force, pressure, velocity, acceleration, position), thermal (temperature, entropy, heat, heat flow), chemical (concentration, composition, reaction rate), radiative (electromagnetic intensity, phase, wavelength, polarization, reflectance, refractive index, transmittance), magnetic (magnetic field strength, magnetic flux density, magnetic moment, permeability), and electrical (voltage, current, charge, resistance, capacitance, polarization).

[0061] As used herein, “actuator” is a device that converts a signal into action. It can generate force to manipulate itself, other mechanical devices, or the surrounding environment in order to perform some useful function.

[0062] As used herein, "transducer" is a device that converts one form of signal or energy into another form. Therefore, the term transducer can be used to include both sensors and actuators.

[0063] As used herein, “nucleotide” is an organic molecule consisting of a nucleoside and a phosphate functional group. Nucleotides function as monomeric units of deoxyribonucleic acid or ribonucleic acid polymers. There are two types of nucleotides (purines and pyrimidines). Specifically, purines are adenine (A) and guanine (G). Specifically, pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA, and U is found in RNA.

[0064] An amino acid is an organic compound that contains both an amino functional group and a carboxylic acid functional group. Amino acids function as the monomeric units of peptides and proteins. Secondary, tertiary, and quaternary structures influence the properties, function, and conformational dynamics of proteins.

[0065] In this specification, the terms “polypeptide,” “protein,” and “peptide” are used synonymously to refer to amino acid chains in which amino acid residues are linked by peptide bonds or modified peptide bonds. Amino acid chains can be of any length, consisting of more than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass their various modified forms. These modified forms may be spontaneously occurring or chemically modified. Examples of modified forms include, but are not limited to, glycosylated, phosphorylated, myristoylated, palmitoylated, ribosylated, and acetylated forms. Modifications also include intramolecular crosslinking and covalent bonding of various moieties such as lipids, flavins, biotin, polyethylene glycol, or derivatives thereof. Furthermore, modifications may include protein cyclization, amino acid chain branching, and protein crosslinking. Additionally, polypeptides may contain amino acids other than the usual 20 amino acids encoded by genes.

[0066] The terms “protein” or “polypeptide” may also include “purified” polypeptides that are substantially isolated from other polypeptides in naturally occurring cells or organisms (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0067] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, the use of "or" is intended to include "and / or" unless the context clearly indicates a different meaning.

[0068] The following descriptive examples illustrate various embodiments and applications of the devices and methods of the present invention. These examples are not intended to be limiting. Other embodiments within the claims of this specification will become apparent to those skilled in the art from considerations herein or from the practice of the invention disclosed herein. This specification, together with the examples, is intended to be considered merely illustrative, and the scope and spirit of the invention are indicated by the appended claims.

[0069] Example 1: BCSR cantilever configuration The physical paradigm of the system of the present invention is two spring elements fixed to one end of a base or reference plane. The first spring is a NEMS cantilever. The second is a crosslinked conformational shift receptor (BCSR), which in this embodiment is DNA for utilizing intrinsic DNA curvature, or "IDC". A crosslinked conformational shift receptor (BCSR) is defined as any structure that generates a vertical force in either direction (upward or downward / pushing or pulling) relative to the cantilever spring by changing its dimensions. In the embodiment of this example, the dimensional change is perpendicular to the cantilever beam plane. The two springs are attached to each other near the first end of their respective physical structures (e.g., at or near the end of the cantilever, and at the functionalized mounting end of the BCSR). The second end of the BCSR is attached to a support structure (i.e., a base or surface fixed to the cantilever spring). Referring to Figures 1A to 1D, the basic concept is that a BCSR contracts by bending (curving) or axially contracting, pulling down the lower surface of the cantilever, when it reacts with or interacts with components in its environment. A complementary pair of BCSRs is designed or selected to achieve a desired shape change so as to induce a measurable force on the attached cantilever spring sensor. Figures 1A and 1C show exemplary embodiments featuring a pre-detection state in which the receptor has not yet associated with the analyte and the cantilever is in a neutral position (e.g., "relaxed"). Figures 1B and 1D show the result of association between the BCSR and the analyte, where the BCSR contracts, pulling the distal end of the cantilever toward the base.

[0070] Changes in the conformational shape of the cantilever can be detected by a variety of techniques, including electrical (piezoresistive, inductive, capacitive, conductive (normally open or normally closed configuration) and optical (laser deflection position), quantum tunneling, fluorescence, or other emission). In an exemplary embodiment, the piezoresistive conversion of the cantilever's displacement / bending / deflection and BCSR react with a complementary component corresponding to spring contraction, resulting in a conformational change perpendicular to the cantilever beam plane.

[0071] Furthermore, the BCSR can also be stretched to achieve displacement from the neutral cantilever position. In addition, the NEMS cantilever can be initially displaced, bent, or flexed by the BCSR, which is base-paired with a complementary nucleotide sequence, thereby relaxing the BCSR or cleaving or altering a specific bond of interest. In this case, the cantilever is restored to its non-perturbed (neutral) position, resulting in a net change in the displacement, bend, or flex of the NEMS cantilever.

[0072] Cantilever displacement can be induced in several different NEMS operating modes. In the "static mode - pre-reaction system," the cantilever does not exhibit a displacement greater than the displacement arising from the total mass of the cantilever and BCSR(s) (i.e., the total mass due to the gravitational field). In the "static mode - post-reaction system," the displacement arises from the contribution of small restoring forces from the cantilever components, where the displacement is mainly due to a change in the conformational shape of the BCSR. In the "dynamic mode," the BCSR is subjected to the relevant force constant k BCSR Modeled as a spring having a certain property, whereas in previous systems, the receptor spring has a high degree of stiffness, holding the cantilever in a permanently displaced or deflected position. Using these different modes of operation, the stiffness and associated forces of the BCSR can be dynamically evaluated according to the present invention's approach, in which the BCSR acts as the center of activity and gives high sensitivity, where changes in its conformation are induced when it interacts, associates, and / or dissociates with one or more analytes.

[0073] Physically and mathematically, the system can be described as having multiple springs in series. In an exemplary embodiment, the device is implemented to have two springs. The BCSR reacts with its complementary pair component and then applies force to the connected force-sensing cantilever spring. The degree of reaction can provide information about the relative pair interaction. However, in a preferred embodiment, if the complementary pair has the required degree of nucleotide base-pair homology, the DNA will have a significant shape change and, consequently, a corresponding force on the cantilever. This property results in a binary detection scheme, i.e., if the resulting complementary pair has a considerable agreement or interaction relationship that results in a conformational change in dimensions, a force is exerted; if there is not a considerable agreement or interaction relationship, little to no force is observed and no conformational change is observed. This is an attractive feature for ensuring a high degree of specificity. Furthermore, one cross-linked conformational shift receptor has enough force to influence the shift of the sensing cantilever spring being measured. This property provides extremely high sensitivity.

[0074] Example 2: Static Mode Operation For a static mode cantilever in which the cantilever and BCSR(sprungs) are in series, the force balance equation for this system is as follows:

number

[0075] In a static system, the acceleration and velocity terms are zero, and the equation is simplified as follows: k eff (y0-y)=F ext (twenty two) Alternatively, Δy = y0 - y, k eff Δy=F ext (twenty three)

[0076] For a system with two series springs, the effective spring constant is the sum of the two springs, where the effective spring constant is as follows: k eff =k c +k BCSSR (twenty four)

[0077] For a more general system description of n springs (a cantilever with n BCSRs), the effective spring constants are as follows:

number

[0078] external force F ext The following applies: F ext =F c +F BCSR (26)

[0079] Equation (23) is as follows: k eff Δy=F c +F BCSR (27) Here, the force applied by the cantilever is F c =k c The force applied by the bridged conformation shape shift receptor is Δy, and F BCSR =k BCSR Δy, where K BCSR This is the force constant of the bridged conformational shape shift receptor.

[0080] In a series spring system, the forces from both springs are different. Since both springs are connected to a common point at the distal end of the NEMS cantilever, the displacements of both springs must be equal, i.e., Δy c =Δy BCSSR (28)

[0081] The displacement of BCSR can be evaluated before and after the reaction with complementary pairs.

[0082] It is important to note that the master equation, equation (28), mathematically demonstrates that it does not depend on the properties of the NEMS cantilever, i.e., the spring constant, length, width, thickness, Young's modulus, and / or mass load. It depends only on the dimensional change of the BCSR that results in a change in displacement / deflection / bending of the NEMS cantilever of equal magnitude.

[0083] Static mode - System before reaction Before reacting with the attached complementary BCSR (ssDNA or RNA), the boundary conditions are adjusted by adjusting the gap between the cantilever tip and the fixed base.

number

number

[0084] The total mass of a NEMS cantilever with BCSR(s) attached is essentially zero.

number

number

[0085] Therefore, before the BCSR reaction, the displacement of the NEMS cantilever is

number

[0086] Static mode - system after reaction After the introduction of a complementary pair, the cantilever spring measures the amount of apparent or effective displacement. If the force of the BCSR is greater than the restoring force of the cantilever, the displacement of the cantilever in either direction is attributable to that displacement of the BCSR. Therefore, in equilibrium,

number

number

[0087]

number

number

number

[0088] Some embodiments utilize a NEMS cantilever with a spatial coordinate definition in Euclidean space. In such a system, the cantilever displacement is defined orthogonally to the cantilever beam in the y-direction. The gap between the mounting point(s) on the cantilever and the BCSR mounting base is on the order of nanometers. The cantilever dimensions include the beam length, L (x-axis), thickness or height, H (y-axis), and width, W (z-axis). The material used to construct the cantilever beam has a Young's modulus, E, moment of inertia, I, and force, F, applied by the bridged conformational shift receptor. BCSR It has.

number

number

[0089] Substituting equation (33) into the cantilever displacement equation (32) and rearranging it, we get the force constant, k c You can obtain this.

number

[0090] Therefore, by selecting the cantilever dimensions (L, H, and W) and the workpiece material (E), the desired cantilever rigidity can be achieved.

[0091] BCSRs can be attached to various points along the cantilever. A BCSR attached to the distal end provides the maximum displacement. When the BCSR attached to the end is compressed to its maximum value, further compression of the cantilever by other BCSRs can also be achieved. The complementary configuration of the BCSRs, i.e., their compression modes, is achieved by their attachment to the cantilever. Details of this attachment are discussed as specific embodiments to provide a clear explanation, but the discussion is not intended to be limited to the following specific examples.

[0092] It should be noted that the geometric design characteristics and fabrication material of the cantilever play only a small role in the cantilever's displacement. Rather, the key is the BCSR, which determines the displacement vector along the line between the two mounting points. This vector does not need to be exactly orthogonal to the cantilever beam, i.e., 90°, but in the event of a configuration that is not orthogonal, a reduction in displacement can be predicted. a If the structure (i.e., the "spring") is stiff and relatively incompressible, and if multiple different BCSRs with different mounting configurations are combined on the cantilever, the displacement cannot exceed the length of the most orthogonally mounted BCSR. When multiple BCSRs are mounted on the cantilever, the preferred physical mounting to the tip is the BCSR whose shortest distance across the gap is orthogonal to the cantilever tip. a This can be achieved by physically moving the tip toward the base mounting plate to connect / attach it. a Due to the physical length constraints, BCSR a It cannot be mounted in other areas along the length of the cantilever. a Install the BCSR to ensure a specific, optimal mounting shape (orthogonal) and mounting position (cantilever tip). a After installation, BCSR b To achieve a different conformational structure, complementary pairs are released or restored to an unresponsive state. The system then becomes BCSR. aIt can then be prepared for detection by reaction with its complementary pair to form a compound.

[0093] The NEMS cantilever can be calibrated for specific signal conversion methods. In some embodiments, a piezoresistive transducer is used. The resistive circuit utilizes a Wheatstone bridge configuration, where all resistors are placed directly on the NEMS cantilever, providing the advantage of self-calibration of thermal drift so that all resistors are in close proximity and therefore in thermal equilibrium. To determine the response gradient, the potential across the Wheatstone bridge is calibrated for a specific resistance value and piezoresistive response as a function of the cantilever displacement.

number

[0094] The magnitude of the displacement can be obtained by measuring the change in potential. However, in embodiments using DNA or RNA, a displacement value of any magnitude is necessary to confirm the complementary pair, i.e., the match in the analyte. Once the potential is detected, a considerable match or interaction relationship can be confirmed.

[0095] Example 3: Dynamic Mode Operation A dynamic modal mathematical model can be derived for a spring attached to a NEMS cantilever to which a BCSR is attached in a fixed structure. Here, the BCSR is somewhat flexible, and the relevant force constant k BCSSR We assume that it can be modeled as a spring with the following characteristics. In static mode operation, the receptor spring is very stiff and holds the cantilever in a permanent displacement or deflection position. The stiffness of the BCSR and the associated forces can be dynamically evaluated. The equations of motion for this system are as follows:

number

number

[0096] k eq Substituting this into equation (37), we obtain the following:

number

[0097] Solving equation (39) to determine the resonant frequency yields the following:

number

number

[0098] If the resonant frequency is determined and the dimensional properties, mass, and Young's modulus of the NEMS cantilever are known, the spring constant of the bridged shape-shift receptor can be calculated (Equation (40)).

[0099] Example 4: Cantilever configuration Various embodiments of the cantilever configuration can be formed using one or a combination of the integrated sensors, where the integrated sensor is configured to form a bridged cantilever shape. As used herein, “integrated sensor” refers to a cantilever equipped with means for measuring the deflection of the cantilever against a support base having a fixed gap bridged by a receptor. Exemplary configurations of a device for detecting analytes in a sample are schematically shown in Figures 1A to 1D. Note that although a single cantilever is shown, multiple cantilevers may be arranged in an array. The device comprises one or more integrated sensors and a detector. Figures 1A and 1C show a neutral configuration when the receptor is not associated with the analyte or a configuration physically dissociated from the analyte, respectively. Integrated sensor 2 is configured to form a bridged cantilever shape including a cantilever 10 with a first surface 12, a second surface 14 on a base 16 (or other stable surface), and a receptor 20 with a receptor recognition portion 22. Each of the first surface 12 and the second surface 14 includes one or more contact points for the attachment of one or more receptors 20. Figures 1B and 1D illustrate the devices of Figures 1A and 1C, respectively, and show the physically analyte-associated configuration when the receptor is associated with the analyte. The first end of the receptor is attached to a contact point on the first surface, and the second end of the receptor is attached to a contact point on the second surface. One or more analytes associate with the receptor via one or more molecular structures on the receptor. Due to conformational changes of the receptor in the presence of one or more bound analytes, the first and second ends of the receptor change from their stationary state. The relative distance and angle between the first and second ends of the receptor differ with and without analyte binding.

[0100] Example 5: Other BCSR cantilever configurations Various neutral or physically dissociated sensor configurations are shown in panels A–E of Figure 2, where the receptor is not associated with the analyte. These non-limiting embodiments illustrate various possible combinations of BCSR and cantilever. Panel A shows a single receptor bridged between a stationary surface and a parallel cantilever. Panel B shows a possible embodiment of multiple receptors bridged between a surface and a parallel cantilever. Panel C shows an exemplary configuration having a single receptor bridged between a surface and two parallel cantilevers. Panel D shows an exemplary device in which the association of the analyte causes stress in the suspended “cantilever” material by having a single receptor bridged between two stationary surfaces. Panel E shows a sensor configuration having a single receptor bridged between a planar oriented surface and a cantilever. Based on the disclosure herein, those skilled in the art can devise various different cantilever-receptor combinations to provide sensors.

[0101] Example 6: BCSR Receptor Polynucleotides, polypeptides, and polysaccharides can all change their conformation in response to changes in their environment, such as binding to a substrate or analyte, changes in salt concentration or pH, and phosphorylation. In addition to these biopolymers, other materials are also known to change their conformation in response to environmental changes.

[0102] In some embodiments, the receptor includes inorganic molecules, organic molecules, polymers, polymer analogs, carbohydrates, carbohydrate analogs, nucleic acids, nucleic acid analogs, proteins, protein analogs, antibodies, antibody analogs, repeats of any one of these molecules, complexes of any one of these molecules, hybridizations of any one of these molecules, or any combination thereof.

[0103] A receptor contains one or more molecular structures that function as binding sites for one or more analytes. Upon binding of one or more analytes, the receptor changes to a different conformation by narrowing, bending, or otherwise altering the gap cross-linked by the BCSR.

[0104] The information contained in the genetic material of living organisms far exceeds simple nucleotide coding sequences. Certain patterned structures of specific nucleotides induce higher-order conformations or curvatures of the genetic material. These structures perform crucial biological functions, including the regulation of gene expression, chromosome packaging, transcription initiation and termination, recombination, DNA replication, and nucleosome positioning.

[0105] One well-known example is the mutation of the DNA double helix structure, which exhibits an intrinsic DNA curvature. Single-stranded DNA (ssDNA) does not exhibit an intrinsic curvature, but the hydrogen bonds between the adenine and thymine nucleic acid bases are not perpendicular to the helical axis, and as a result, when bound to an analyte with changes in the composition, position, and length of a certain nucleotide sequence, it can induce an intrinsic bending of the axis. For example, the end-to-end distance of an 85 nm double-stranded DNA (dsDNA) helix can be shortened to 65 nm or more.

[0106] Figure 4 provides a sample plot of the sequence-dependent spatial orbitals of the DNA double helix and the distribution of intrinsic curvature along the DNA molecule. Typically, an adjacent wedge model is implemented to compute the overall DNA structure using local helical parameters (i.e., dinucleotide twist, slope, and rotation angle). Figure 4 shows two peak curvature regions of approximately 4,600 and 26,000 along the adenovirus genome, indicated by arrows.

[0107] In some embodiments, the receptor comprises nucleic acid, nucleic acid analog, double-stranded DNA (dsDNA), dsDNA analog, single-stranded DNA (ssDNA), ssDNA analog, or peptide nucleic acid.

[0108] In some of these embodiments, the receptor is ssDNA. In other embodiments, the receptor may be a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

[0109] In some embodiments, the receptor has a length of 50 to 1,000 nm. Those skilled in the art can design / manufacture the receptor length to optimize performance for a particular application.

[0110] Another well-known example of conformational change is the two conformational isoforms of prion proteins. The cellular isoform of the prion protein is the alpha-helix-rich prion protein (PrPC), while the prion isoform is the beta-structure-rich insoluble conformational isomer (PrPSc).

[0111] Example 7: BCSR response to analytes The analyte can associate with receptors on the cantilever, potentially causing conformational changes in the receptors. The analyte and receptors are held together through non-covalent interactions, such as electrostatic interactions, van der Waals interactions, π-effect interactions, and hydrophobic and hydrophilic interactions.

[0112] In some embodiments, the analytes include inorganic molecules, organic molecules, polymers, polymer analogs, carbon nanotubes, carbohydrates, carbohydrate analogs, nucleic acids, nucleic acid analogs, proteins, protein analogs, antibodies, antibody analogs, repeats of any one of these molecules, complexes of any one of these molecules, hybridizations of any one of these molecules, or any combination thereof.

[0113] In other embodiments, the analyte includes complementary nucleic acids, complementary nucleic acid analogs, complementary dsDNA, complementary dsDNA analogs, complementary ssDNA, complementary ssDNA analogs, complementary RNA, or complementary RNA analogs.

[0114] The methods and devices described herein are not limited to the use of any particular sample type. Examples of samples that can be tested using the devices and methods of the present invention include tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal specimens, nasopharyngeal fluid, urine, feces, tissue, blood, plasma, serum, cell cultures, body fluids, tissue biopsies, apocrine fluid, or eccrine fluid. Sample types and sources may further include forensic, aerosol, soil, water, food, components, raw materials, in-process, by-products, finished products, or quality control samples.

[0115] In some embodiments, the sample may be a virus, bacteria, phage, yeast, mycoplasma, fungus, human cell, animal cell, plant cell, insect cell, or any combination thereof. The approach of the present invention has no molecular limitations on its ability to detect any virus (bacteria, fungi, etc.), regardless of whether it is a nucleic acid, protein, or other composition.

[0116] The association of BCSR with the analyte can induce various conformational changes that cause changes in the relative positions of the cantilever and the reference. Panels A–F of Figure 3 schematically illustrate the relative motion of an exemplary sensor having a single bridged conformational shift receptor between itself and a cantilever parallel to the surface. Panel A of Figure 3 shows an exemplary sensor in which the single bridged conformational shift receptor is physically dissociated from the analyte (i.e., neutral configuration). Panel B of Figure 3 shows an exemplary sensor in which the single bridged conformational shift receptor is physically associated with the analyte, demonstrating a tensile action on the cantilever. Panel C of Figure 3 shows an exemplary sensor in which the single bridged conformational shift receptor is physically associated with the analyte, demonstrating a compressive action on the cantilever. Panel D of Figure 3 shows an exemplary sensor in which the single bridged conformational shift receptor is physically associated with the analyte, demonstrating a rotational action on the cantilever. Panel E of Figure 3 shows an exemplary sensor in which a single cross-linked conformation shift receptor is physically associated with the analyte, illustrating a torsional action on the cantilever. Panel F of Figure 3 shows an exemplary sensor in which a single cross-linked conformation shift receptor is physically associated with the analyte, illustrating a combined tensile and torsional action on the cantilever. A wide variety of substitutions are possible beyond the exemplary embodiments. To provide another alternative configuration, the plane orientation shown in Panel E of Figure 2 can be modified so that the BCSR causes relative tensile, compressive, rotational, and / or torsional forces between the cantilever and the support surface. Those skilled in the art can modify the receptor configuration for specific applications without unnecessary experimentation.

[0117] In most embodiments, the interaction, association, or dissociation of the receptor with one or more analytes induces a change in the relative position of the first surface (i.e., the base or reference plane) and the second surface (i.e., the cantilever). In some embodiments, the distance between the first and second surfaces is 1 to 1,000 nm.

[0118] Example 8: Resettable BCSR Referring to Figure 5A, the association of the BCSR 52 with the target analyte 54 can cause the intrinsic curvature or axial contraction of the crosslinked receptor 52, the bending of the piezoresistive cantilever 50 (as shown in step 2), and consequently, a change in resistance. In some embodiments, the device is discarded after step 2 after being used once. In other embodiments, the receptor 52 and analyte 54 may be reset by any means of dissociating them, including thermal, electrical, electrochemical, or chemical methods. The analyte may be fragmented, destroyed, or removed by flushing or evacuating the test chamber. In some embodiments, the test chamber is flushed with a relatively alkaline buffer solution to denature the dinucleotide complex and separate the analyte from the receptor (e.g., step 3). Dissociation of the analyte from the BCSR returns the receptor to its original conformation and relaxes the piezoresistive cantilever, thus causing a change in resistance as shown in Figure 5B.

[0119] Example 9: Signal Conversion The function of piezoresistive materials is well known to those skilled in the art. In short, in piezoresistive materials, changes in physical properties are converted into changes in electrophysical properties. In the context of this cantilever sensor, a flexible piezoresistive material is bonded to or coated onto a substrate surface configured to deform when force is applied to the material. When the substrate surface is bent, the crystalline structure of the piezoresistive material is also subjected to deformation or stress, thereby changing its resistance properties. The sensitivity coefficient is determined as the ratio of the change in stress to the change in resistance (i.e., stress / resistance). It is important to note that the conversion material includes the piezoresistive coating on the substrate surface, not the substrate surface itself.

[0120] The deflection / displacement of the first and / or second surface can be measured by directly contacting a transducer element with either the first or second surface. The signal conversion via the transducer is related to changes in electrophysical properties (V, I, R), changes in the magnitude of the electromagnetic amplitude, changes in the EM field strength, changes in the EM frequency, changes in the EM position vector, etc., thus measuring changes in physical properties. The magnitude of the converted signal is proportional to the sensitivity coefficient (slope). In some embodiments, a thin coating of flexible piezoresistive material is bonded to the cantilever surface. The BCSR bends the cantilever surface and simultaneously bends / deforms the crystal structure of the piezoresistive material. The deformation or stress of the crystal structure changes the resistance properties. The sensitivity coefficient is the change in stress divided by the change in resistance (slope; δ displacement / δ resistance, or δD / δR).

[0121] The conversion material is a patterned piezoresistive coating on the surface. Mathematically, Δ-displacement = (δD / δR)Δ-resistance, or similarly, Δ-resistance = (δR / δD)Δ-displacement.

[0122] Referring to Figure 6A, a MEMS cantilever-based sensor assembly 60 is schematically shown. The cantilever arm 62 is formed from silicon or silicon-on-insulator (SOI) coated with a piezoresistive material to generate a detectable electrical signal when a force is applied to the arm. In some embodiments, the underside of the cantilever may be coated with gold to facilitate bio-attachment to a BCSR(s). The proximal end of the arm 62 is bonded to a support base 64 on which an electronic circuit is patterned. Note that the illustrated interconnection pattern is merely representative and does not represent the layout of electrical connections within the device. (See Example 7 below: A Wheatstone bridge circuit used for measuring resistance changes.) As further described below, an electrical signal (e.g., V) is transmitted to and from an external device. in , V out , V ccFor the purpose of exchanging signals, a conductor 66 extends at least partially along the length of the cantilever arm 62 and is embedded in or otherwise connected to the piezoresistive material within the cantilever, to communicate signals generated within the piezoresistive material to various conductors and bond pads 68 on the support base 64. The base portion 64 is typically bonded to a printed circuit board (not shown) using conventional wire bonding techniques used to form the connection. The base portion 64 and the PCB may be sealed in a package or epoxy sealant for protection and to facilitate connection to a socket or multi-pin connector. As previously mentioned, in most embodiments, the BCSR is typically mounted on the underside of the distal end of the cantilever arm 62 and on the upper surface of the support base 64 to bridge the gap.

[0123] In some embodiments, the fabrication of MEMS piezo-resistive cantilever assemblies may utilize conventional semiconductor manufacturing techniques, which may include standard photolithography patterning steps, chemical and plasma etching, electron beam writing, thermal diffusion and / or ion implantation for doping, chemical deposition, sputtering, and the like.

[0124] Figure 6C schematically illustrates an exemplary processing sequence for manufacturing a cantilever arm on a silicon-on-insulator (SOI) wafer according to one embodiment. Based on this exemplary sequence, it will be readily apparent to those skilled in the art that modifications of the process sequence can be made without departing from the general principles of the present invention's approach, which is to provide a precise cantilever with reproducible dimensions and performance.

[0125] The starting substrate is a silicon-on-insulator (SOI) wafer, i.e., silicon and oxide (SiO2) on a handle layer. Exemplary starting wafer layer thicknesses can be on the order of 10 μm, 1 μm, and 400 μm, respectively. The following steps for fabricating a cantilever structure according to one embodiment of the approach of the present invention are labeled (a) to (m) in Figure 6C. Various steps utilize conventional semiconductor processing techniques well known in the art and are therefore not described in detail. (a) A thermal oxide (SiO2) (approximately 500 nm) is grown on a wafer. (b) After patterning the structure, the oxide is etched to expose the handle layer. (c) N-type doping and diffusion into exposed structures. (d) Remove surface oxides. (e) Thermal oxidation (approximately 100 nm). (f) Patterning and oxide etching are performed to open up the structure to define the piezoresistive within the Si layer, and then P-type doping / diffusion is carried out. (An ion implant may be used in this step.) The exemplary shape of the piezoresistive is approximately 10 μm wide × 50 μm long (twice in series) with a diffusion depth of approximately 0.25–0.5 μm, which is estimated depending on the doping concentration. (g) Oxide etching is performed to grow thermal oxide (approximately 100 nm). (h) Patterning and etching of oxides are performed to expose the contact window. (i) Deposit contact metallization (Al / 1%Si) and etch it onto the pattern. (j) Deposit Si3N4 ("SiN") passivation using plasma-enhanced chemical vapor deposition (PECVD), then pattern and etch. (k) Pattern the cantilever tip with gold or Ti / Au for BCSR mounting. (l) Etch the cantilever and dicing street (see steps (s) and (t) below). (m) Etch the handle wafer to isolate the device (see step (u) below).

[0126] The resulting device corresponds to a cantilever arm (i.e., one of two surfaces to which one end of the BCSR(or BCSR) is attached). Illustrative dimensions of some embodiments of the cantilever are on the order of 400–450 μm in length, 100–115 μm in width, and approximately 3.8–5 μm in thickness. The cantilever's resonance will be on the order of 15–40 kHz. This cantilever is permanently mounted to a raised pedestal that is mounted orthogonally to a rigid surface (i.e., a mounting base) forming a second component of the sensor device. The fabrication of the mounting base is described below.

[0127] Figure 6D schematically illustrates the process flow for fabricating the second component of the sensor (i.e., the mount base) and assembling the cantilever structure onto the base. The base provides electrical connections between the piezoresistive element on the cantilever structure and the external interconnect. The following steps for fabricating the base structure according to one embodiment of the approach of the present invention are labeled (n) to (u) in Figure 6D. The following steps utilize conventional semiconductor processing techniques well known in the art and are therefore not described in detail. (n) Starting with an SOI wafer, a layer of thermal oxide is grown on the Si surface. (o) Deposit polysilicon onto an oxide surface (e.g., PECVD) and pattern it to define a pedestal for supporting the cantilever. (p) Pattern and etch the oxide to form an opening for connection to the substrate ground. (q) Ti / Au is deposited on the top of the wafer and patterned to define the electrical contacts and the gold (or other material) area for BCSR mounting. (r) The top surface of the cantilever on the handle wafer is positioned relative to the base wafer so that the proximal end of the cantilever is aligned with the polySi pedestal and the gold region at the distal end of the cantilever is aligned with the gold region on the base. (Alignment guides are patterned on both wafers according to standard semiconductor processing techniques.) Once aligned, the assembly undergoes heat treatment for gold-silicon eutectic formation. As is well known, Au-Si eutectic formation occurs at approximately 360–370°C. Exemplary temperatures for bond formation are on the order of 410–470°C. (s) Etch the cantilever and dicing street. (t) Carrier mounting and wafer thin backing for separating the handle layer from the cantilever. (u) Final disconnection of the completed device.

[0128] The bond pad formed in step (q) is electrically connected to the corresponding connector on the PCB to provide an external connection.

[0129] The key steps in the process include steps (o), (q), and (r), the combination of which enables the formation of a reproducible and clearly defined gap between the bottom surface of the cantilever and the top surface of the base. Specifically, the gap between the contact region near the distal end of the cantilever (labeled "gold contact #1" in Figure 6D) and the gold contact region directly below the distal end of the cantilever (labeled "gold contact #2" in the figure) is a critical structural component of the sensor device of the present invention. These gold contact surfaces are approximately 0.1 nm thick. 2 (For example, the approximate diameter of a single receptor molecule) ~1,000 nm 2The above range is acceptable. The resulting structure defines a gold-plated / coated gap between two surfaces that serve as attachment points for the molecular sub-attachment ends. As previously stated, in the described embodiments, gold is used as the material for the BCSR contact points, but the contact area material may be a variety of other materials such as silicon, silicon dioxide, glass, quartz, polymers, platinum, titanium, tin, aluminum, nickel, copper, and iron. The base surface is a rigid structure that does not bend or deform, while the cantilever surface is flexible and can be bent or flexed. This physical configuration is a NEMS device that functions as a detection unit. The height of the polysilicon pedestal formed in step (o) can be controlled to tight tolerances of a few nanometers using well-known deposition techniques. The thickness of the selected pedestal, combined with the thickness of the gold contact (step (q)) and the eutectic junction formed in step (r) (which can be expected to be very thin (on the order of less than 200 Å)), defines the gap. The gap can vary from 5 nm to several hundred microns by controlling the thickness, primarily defined by steps (o), (q), and (r). This integrated cantilever structure provides a basis for further modification and adaptation to attach / bond single or multiple molecular parts to bridge the gap.

[0130] Figure 6E provides a diagram of the integrated sensor 100 of the present invention according to one embodiment manufactured according to the process flow shown in Figures 6C and 6D. (It should be noted that this diagram is not to scale; rather, the dimensions are greatly exaggerated to facilitate the illustration of key components of the integrated sensor of the present invention, including the placement of receptors in the gaps.) The legend is a legend for the materials used in the manufacture of the sensor 100.

[0131] This ability of the present invention's approach to provide precise and repeatable control over gap dimensions overcomes the shortcomings of previous efforts to manufacture reliable cantilever sensing devices for use in crosslinking sensing applications.

[0132] In some embodiments, the ends of a receptor containing ssDNA are attached to or chemically bonded to two gold contact regions using a thio-gold chemical. Other attachment chemicals are available depending on the molecular structure of the crosslinking portion. This chemical has been proven to have the physical strength necessary to maintain the attachment during changes in the shape of the molecular structure. The NEMS integrated cantilever unit is designed and manufactured to a desired force constant that is relatively weak compared to the forces exerted by the structure of the molecular portion. This configuration provides the necessary conditions for the cantilever surface to be displaced during changes in the shape configuration of the molecular portion (e.g., dsDNA). When a single strand (ssDNA) is attached at each end between the NEMS cantilever and the surface fixed to the integrated NEMS cantilever, it is relatively linear and does not exert much force to bend or displace a flexible cantilever structure that is normally in position or neutral (i.e., flat). When highly complementary or matching ssDNA binds / reacts with the attached ssDNA element, it forms a corresponding dsDNA helical structure. The dsDNA molecule is specifically selected through modeling and experimental validation to have a highly curved structure. The newly formed curved structure has a force constant much larger than that of the MEMS cantilever, causing it to bend or flex. The displacement is measured by a mounted piezoresistive(s) positioned on or integrated into the surface of the NEMS cantilever. The piezoresistive(s) are positioned on the cantilever via a Wheatstone bridge configuration at a location that achieves the greatest degree of deformation (i.e., near the pedestal mount). The deformation of the piezoresistive material causes a change in its crystal structure, thereby inducing a change in resistance measured by the potential change across the Wheatstone bridge design.

[0133] Example 10: Detection System Figure 7 schematically shows a prototype detection system 70 used for preliminary testing and evaluation of the present invention's approach to detecting analytes using an integrated sensor. This embodiment operates using the principle of a Wheatstone bridge, which is a simple series-parallel arrangement of two resistors connected between voltage supply terminals, where the resistors are piezoresistive coatings on a cantilever arm 62 that are converted into analog signals.

[0134] A voltage source 72 provides voltage to the Wheatstone bridge defined by PCB 64 and arm 62. Figure 6B provides a diagram of a half-active full Wheatstone bridge that may be used in several embodiments. A sensor assembly 60 may be plugged into a connector socket 63 that provides connections to the voltage source 72 and preamplifier 74, which receive signals from the sensor assembly 60 to measure voltage changes from the Wheatstone bridge. The measured voltage changes are converted from analog to digital by an analog-to-digital converter (ADC) 76. The preamplifier 74 provides a 100 gain amplified signal from the Wheatstone bridge to the ADC, which is then input to a system computer controller 78 that receives the measurement signal and, at the same time as receiving the measurement signal, provides control signals for the operation of the system 70, including the control, operation, and movement of an optional flow cell assembly 80, which is further described below with reference to Figures 8A-8B. The computer controller displays the data graphically on the screen as a function of the cantilever output voltage as a function of time. Its software was developed using NI-DAQmx (version 21.3). The amplified output signal was 20 nm / volt.

[0135] Flow cell controls that may be provided by the computer controller 78 include XYZ directional control for a precision multi-axis positioning stage 79 used to position the sensor assembly within the test well or chamber, flow parameters, and valves for introducing the analyte and / or flushing / rinsing the flow cell. The computer controller 78 may also include memory for storing results and links to external memory and / or communication systems for reporting measurement results.

[0136] A magnified optical imaging system was designed, manufactured, and tested to visually observe the cantilever positioning on the slide base and the bending of the MEMS cantilever. A camera captured images / videos of the magnified cantilever, projected them onto a computer screen, and / or transmitted the visual information to the computer.

[0137] In the test setup, the positioning stage 79 was a piezo bending stage mounted beneath the cantilever to adjust the X, Y, and Z positions of the gold-coated surface from which the receptor bridged to the cantilever tip. A piezo bending driver unit controlled the precise positioning of the stage to allow nanometer-scale control of the gap distance between the gold surfaces. To achieve a cost-effective and flex-sensitive cantilever system, the piezo bending stage was selected to have an open loop with X, Y, and Z moment components of 267 nm / V. For each bolt applied to each channel, the piezo bending element moved 267 nm over the entire length of a 20 μm travel range (up to 75 volts). The piezo bending stage and all other dynamic moving components were mounted on an 18-inch × 24-inch breadboard optical bench with vibration-damping feet. A slide holding fixture was mounted directly onto the piezo bending stage. A gold-coated glass slide was mounted on a slide holder mount to provide the base bioreceptor mounting area. To adjust the gap between the cantilever and the base, the piezo bending stage was raised or lowered as needed during various experimental processes. The piezo bending stage may be operated manually using a control knob on the piezo bending control module, or it may be connected to both ports via a USB cable via a computer software program and GUI running on a computer. The cantilever mounting fixture consisted of multiple components, a raised mounting fixing stage, and a magnetic cantilever mounting head for easy removal and replacement. The cantilever mounting head allowed the PCB of the MEMS cantilever to be secured to it via a screw-adjustable clamp device. Furthermore, a microscope video camera was mounted on an optical breadboard to visually inspect the alignment and bending of the MEMS cantilever during the execution of experiments.

[0138] In the experimental setup, an amplification circuit supplied 2.048 volts to a Wheatstone bridge. This circuit provided a positive potential difference when the MEMS cantilever was bent upward and a negative potential difference when it was bent or pulled downward. Two of the piezoresistives were placed at the base of the cantilever, and the other two were placed near the base of the cantilever on the stationary section of the MEMS device. Two of the piezoresistives were placed at the bottom of the cantilever to experience the physical dimensional changes of expansion (upward bending) and compression (downward bending). This physical change of the piezoresistive material on the cantilever provided a means of conversion for detecting the degree of bending in the MEMS cantilever. Connector socket 63 allowed for rapid replacement of the sensor assembly.

[0139] To provide positive control from the MEMS cantilever to the computer output display, the stage was operated upward with a vernier control knob (first coarse control, then fine control) until a gold-coated glass slide touched the tip of the MEMS cantilever. The MEMS cantilever voltage output was monitored as the tip was about to engage and adjusted to a baseline offset voltage representing zero deflection. The mounted MEMS cantilever was then deflected upward by moving the piezo bending stage upward and measuring the voltage opportunity on the computer data output window. The MEMS cantilever voltage changes were monitored to check for appropriate deflection changes (i.e., voltage magnitude and voltage direction (+ / -)) during upward and downward piezo bending controller sweeps. This process ensured that the MEMS cantilever and piezo bending stage responded as desired before the receptor was mounted and the detection experiment was performed.

[0140] The preliminary tests involved bending the cantilever up and down with a stage controller to examine the data acquisition (DAQ) output voltage. The stage was positioned under a fixed cantilever using a vernier dial to a position where the cantilever was visually observed to engage with the slide, and then the stage was slightly retracted to disengage the cantilever. Next, the cantilever was re-engaged and the Z-direction was adjusted upward with a piezo bending control system to bend it over a range of values. A camera mounted above the cantilever recorded the original cantilever position before bending. Focus and defocus occurred as the position of the cantilever tip was displaced. Bending the cantilever downward was achieved by adjusting the cantilever tip under a suspended microscope coverslip fixed to the stage. The coverslip pushed the tip downward when lowered over the cantilever. The DAQ response at the applied stage controller voltage versus voltage, and its conversion to nanometers (1V=10nm), were then plotted. When the cantilever was bent upward, the DAQ voltage increased. Conversely, when the cantilever was bent downward, the DAQ voltage decreased. This was more similar to the response expected when the IDC curvature of a bridged receptor pushes the cantilever downward toward the stage.

[0141] Example 11: Optional Flow Cell Assembly Referring to Figures 8A and 8B, the flow cell assembly 80 provides an optional approach for precisely and repeatedly positioning a cantilever-based sensor assembly within a reaction chamber or flow cell, allowing the analyte to interact with the receptor. The flow cell assembly 80 has a base plate 85 with a laminated structure having capillary channels 87 extending between the layers. The channels 87 provide fluid connections between the test well 86 and connectors 82 and 84 extending from the top surface of the plate 85. Connectors 82 and 84 are configured to connect to standard laboratory tubing connectors for supplying fluid to the test well 86. Connector 82 provides fluid inflow and outflow for flushing the test well and sensor system. The fluid used for flushing may be a sterile solution or a solution selected to reset the sensor to dissociate the receptor and analyte (e.g., an alkaline buffer solution). Connector 84 provides introduction of the analytical fluid into the test well via a pipette, dropper, or other applicator.

[0142] Figure 9 is a flowchart illustrating the steps of an exemplary pathogen detection process using the system of Figure 7 with an optional flow cell assembly. In step 91, a BCSR is fabricated using a double-stranded semi-modified DNA bioreceptor in which one strand is modified at both the 5' and 3' ends and the other strand is unmodified. The BCSR is used to bridge the gap between the cantilever tip and the floor of the test well 86. (Note that when using the integrated sensor 100 (Figure 6E), the BCSR bridge bridges the gap between the cantilever tip and the support base.) In step 92, the flow cell assembly is lifted by a computer controller providing a command to the positioning stage 79 to create a 150 nm gap between the cantilever tip and the test well floor. The dsDNA BCSR is then attached to the cantilever tip and the well floor in step 93. In step 94, the positioning stage is controlled to lower the flow cell assembly by approximately 5 nm to induce cantilever deflection. Note that the exact distance is not critical. This initial cantilever deflection is used to confirm the success of the initial bridge formation, and system diagnostics are used to confirm the integrity of the bridge during testing. Next, the dsDNA is denatured in step 95 by introducing a chaotropic agent or other denaturing fluid through tube 88 and flow channel connector 82. This permanently attaches the modified strands across the gap between the cantilever tip and the well floor. The unmodified receptor complement is then flushed out of the test well in step 96. In this sequence, steps 91-96 can be classified as a sequence known as “biobridge construction”.

[0143] The following steps constitute the "pathogen detection" sequence. In step 97, a sample containing suspected receptor complement (e.g., the pathogen being tested) is introduced into the test well through sample port 84. In step 98, if the sample contains suspected receptor complement, the dsDNA contracts or bends as a result of IDC, pulling down the cantilever. Due to the bending of the cantilever, the piezoresistive element changes resistance, which is detected by the detection assembly and communicated to the computer controller for outputting the test result (i.e., a positive test), and stored in computer memory or external memory. If the sample does not contain suspected receptor complement, no bending occurs, and a negative result is reported by the computer controller.

[0144] Referring to Figure 8C, exposing the integrated sensor 100 to the analyte for pathogen detection can be achieved by one of many different methods, such as depositing the sensor 100 at the bottom of the test well 102 and / or fixing the sensor 100 at the bottom. The test well 102 may be a single container or one well in a well array in a multi-well plate, as used for high-throughput screening of samples. The test well 102 may be filled with a sample preparation reagent into which the analyte sample is introduced. It should also be noted that there are no restrictions on the size or dimensions of the reaction chamber or test well, and any number of integrated sensor assemblies 100 may be placed in the reaction chamber.

[0145] The electrical connector 110 provides a connection between the conductive pads on each sensor 100 and the system electronics, as described with reference to Figure 7. Although not shown, the bond pads of the integrated sensors may be connected via, for example, die attach, ball wedge bond, wire bond, or similar, and the connected PCB may be connected to a multi-contact socket, multi-pin plug, flex cable pinout, or similar structure to facilitate quick electrical connection and disconnection of the sensors to the system electronics. The analyte 106 can be introduced into the test well 102 by a dropper 104, pipette, or similar applicator. In some applications, the test well may be a chamber, canister, or cuvette 120 into which an aerosol sample 122 can be introduced to expose the sensor 100, rather than a container configured to hold a liquid, as shown in the example in Figure 8D. (Note that electrical links to external devices are not shown.) The aerosol sample can be introduced using an approach similar to that used in breath analyzers. The underlying measurement principles differ between the principle for detecting alcohol (by utilizing the oxidation of ethanol to acetic acid) using a conventional breath analyzer and the principle for detecting pathogens or other analytes by utilizing the interaction with the receptor of the integrated sensor of the present invention. However, the exposure mechanism is similar insofar as the subject can simply exhale air 122 into a mouthpiece 124 connected to a test chamber 120 in order to enable direct detection of the presence of the target by the sensor 100.

[0146] The association between the target analyte in the sample and the biobridge receptor induces a structural change in the cross-linked molecule (BCSR), which causes the cantilever to flex. During flexing, the piezoresistive element within the cantilever induces a change in output voltage, generating a detection signal. The test well may be of any shape or size, and there is no limit to the number of biobridged cantilevers available for testing. Alternative structures and approaches for exposing the sensor 100 to the analyte under test will readily become apparent to those skilled in the art.

[0147] Example 12: BCSR sample processing reagent (SPR) Sample preparation reagents (SPRs) for detecting analytes in a sample are provided herein. SPRs comprise buffers, enzyme activity inhibitors, denaturants, low ionic strength detergents, and oxidizing agents. SPRs allow receptors within an integrated sensor to associate with one or more analytes in the sample. Furthermore, SPRs allow the resulting conformational changes of the receptors to induce changes in the cross-linked cantilever shape.

[0148] Buffers in biological systems maintain intracellular and extracellular pH within a very narrow range, resisting pH changes in the presence of internal and external influences. pH in biological systems controls the solubility, biological function, and chemical reactivity of biomaterials. Buffers maintain a pH that allows analyte nucleic acids to dissociate from their native complement or intrinsic secondary structure. Non-limiting examples of SPR buffers include phosphates, MES, bistrispropane, TES, histidine, HEPES, DIPSO, MOBS, TAPSO, Tris, Trizma, HEPPSO, POPSO, TEA, EPPS, trichine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CABS, and CABS. In various embodiments, the SPR is pH 7–11.

[0149] Detergents are surfactants with an amphiphilic structure, where each molecule has a hydrophilic (polar) head and a long hydrophobic (nonpolar) tail. The dual nature of detergents promotes the mixing of hydrophobic compounds and water in biological samples, for example.

[0150] SPR detergents can be anionic detergents, cationic detergents, amphoteric detergents, defoaming detergents, or any combination thereof.

[0151] Low ionic strength detergents can disrupt cell wall structures, allowing analytes to be released from cells.

[0152] In some embodiments, the detergent comprises Triton X-100, TWEEN-20, NP-40, or a Brij-surfactant.

[0153] An oxidizing agent is a chemical species that tends to oxidize other substances, cause them to lose electrons, and increase their oxidation state. The oxidizing agent in SPR causes controlled oxidative fragmentation of the analyte.

[0154] In some embodiments, the oxidizing agent comprises oxygen, ozone, a peroxide compound, a nitrate compound, a nitrite compound, a chlorine compound, a perchlorate compound, a chlorate compound, a chlorite compound, a hypochlorite compound, a bromine compound, an iodine compound, a persulfate compound, a permanganate compound, a chromium compound, a chromate compound, a perborate compound, a bismuthate compound, a copper compound, an iron compound, a cerium compound, a lead compound, a silver compound, a rhenium compound, a hydroxylamine compound, a sulfur oxide compound, an osmium oxide compound, an N-oxide, a quinone compound, an amide compound, an imide compound, a cyanate compound, a saccharin compound, a selenate compound, a perhenate compound, propyl gallate, or any combination thereof.

[0155] In certain embodiments, the oxidizing agent comprises a peroxide compound, a nitrate compound, a permanganate compound, or any combination thereof.

[0156] In some embodiments, an enzyme activity inhibitor is present in the SPR. Non-limiting examples of enzyme activity inhibitors useful for SPR include chelating agents, RNase inhibitors, DNase inhibitors, protease inhibitors, or any combination thereof.

[0157] A chelating agent is a compound that reacts with metal ions to form stable water-soluble metal complexes. The presence of a chelating agent in a biological sample protects nucleic acids from enzymatic degradation by removing metal ions. Also, chelating agents reduce the interaction between proteins and nucleic acids and thus increase the nucleic acid extraction efficiency in a biological sample. Non-limiting examples of SPR chelating agents include EDTA, EGTA, HEDTA, NTA, and TEA.

[0158] RNase inhibitors, DNase inhibitors, and protease inhibitors may also be present in SPR to prevent the analyte from degrading during the process of using SPR to detect the analyte in the sample.

[0159] In further embodiments, the SPR further comprises a chaotropic agent. The chaotropic agent disrupts the hydrogen bonding network in aqueous solution, destabilizing the native state of macromolecules (e.g., proteins, nucleic acids) in solution. The chaotropic agent denatures nucleic acid-associated proteins and thus weakens the hydrophobic interactions between the nucleic acid-associated proteins and nucleic acids. The chaotropic agent dissociates nucleic acids from nucleic acid-associated proteins. Non-limiting examples of chaotropic agents include guanidinium thiocyanate, guanidine, urea, and thiourea.

[0160] Those skilled in the art can understand and optimize the individual components of a sample preparation reagent (SPR) used to detect analytes in a sample.

[0161] Example 13: Detection of SARS-CoV-2 by bending of a biocantilever To enable attachment of the cantilever sensor to the gold surface, the 5' and 3' ends of candidate receptor DNA were chemically modified. Specifically, the 5'-phosphate was thiolated via a standard alkaline phosphatase and T4 kinase reaction using ATPγS. The 3' end was modified via a T4-ligase reaction to the 3'-thiol-modified oligonucleotide. The success of the thiol modification was demonstrated by electrophoretic resolution of purified unmodified and thiol-modified receptor DNA. In the absence of an appropriate concentration of reducing agent (i.e., DTT or TCEP), the thiol-modified receptor forms disulfide-linked polymers. Prior to bioassembly, the thiol-modified DNA ends were reduced with TCEP to remove the thiol-thiol-linked polymers.

[0162] The cantilever body contains a piezoresistive material whose conductivity or resistance changes as a function of the cantilever's deflection. The cantilever tip and the support surface below the tip are gold-coated to allow bioattachment to thiol-modified receptors. Twenty silicon piezoresistive MEMS cantilevers with various shapes and physical properties (e.g., material, length, force constant, and Wheatstone bridge resistance values) were fabricated. Initially, two different lengths of 400 nm and 450 nm, as well as two different tip designs, were selected. The shorter cantilevers had sharp Si tips, while the longer ones were tipless. Figure 10 is a plot of experimental details when various samples were tested with the two cantilevers.

[0163] For the initial test, the module was mounted on a silicon wafer using double-sided Kapton tape. A second piece of double-sided Kapton tape was used to shadow mask the sensor tip and for mounting. The plastic protectant was later removed only on the outside of the small (approximately 1 inch) piece of tape. The wafer was placed on a substrate holder and loaded into the electron beam evaporation system. Substrate rotation was turned off due to the distance from the masking tape to the cantilever tip, which would lead to gold deposition beneath the tape. After depositing Ti / Au stacks (5 / 50 nm) at 1 A / s and 2 A / s respectively, the sample and tape were removed.

[0164] The BCSR bioassembly and detection approach involved a series of steps to detect the presence of a complementary target molecule using a receptor-attached cantilever. This approach followed a general sequence shown in Figure 9. First, the gold surfaces of the cantilever and support base were prepared, and then a gap was formed between them. Voltage measurements were performed using a Data Acquisition (DAQ) system. Next, the gap was crosslinked by introducing the receptor, and an increase in the DAQ voltage was observed as the gap size increased, providing evidence of crosslinking. Unbound complementary strands were removed by flushing the system, demonstrating the formation of a single-stranded DNA bridge, resulting in a change in the DAQ voltage. Before bioassembly, it was essential to confirm that the voltage was different from the voltage of the uncrosslinked gap. In the detection sequence, a sample containing the complementary target molecule was introduced. The DAQ voltage was measured as an indicator of cantilever deflection due to target molecule binding. Finally, these steps were repeated for further detection tests.

[0165] To evaluate the sensitivity and specificity of BCSR component assemblies, nine different samples were evaluated with DNA receptor 008. The nine samples were as follows: (1) Positive control - complement: A positive control sample containing approximately 100 copies of 100% complementary DNA in the sample preparation reagent (SPR) solution. (2) Negative control - non-complement: A negative control sample containing approximately 100 copies of 40% complementary DNA in the SPR solution. (3) COVID-19 RNA in SPR: Approximately 1000 copies of genomic RNA from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020 (ATCC vr-1986d) were mixed with the SPR solution. (4) COVID-19 virus in SPR: Approximately 1000 copies of heat-inactivated SARS-CoV-2 mutant B.1.1.7 (ATCC vr-3326hk) were mixed with the SPR solution. (5) Sputum in SPR: Human sputum was diluted 100-fold in SPR. (6) COVID-19 RNA in sputum-SPR: Approximately 1000 copies of genomic RNA from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020 (ATCC vr-1986d) in the sputum-SPR solution. (7) COVID-19 virus in sputum-SPR: Approximately 1000 copies of heat-inactivated SARS-CoV-2 mutant B.1.1.7 (ATCC vr-3326hk) were mixed with the SPR solution. (8) Non-COVID RNA in sputum-SPR: Approximately 1000 copies of genomic RNA from human beta-coronavirus OC43 (ATCC VR-1558D) in the sputum-SPR solution. (9) Non-COVID viruses in sputum-SPR: Approximately 1000 copies of human betacoronavirus OC43 (ATCC VR-1558) in sputum-SPR solution.

[0166] Figure 10 is a bar graph showing the deflection of the BCSR biocantilever in nanometers (nm) when the nine samples described above were detected. The nine samples, differing in the presence or absence of COVID RNA, were tested for BCSR biocantilever deflection in nanometers (nm) in 10 replicates. The results showed that positive samples containing COVID RNA produced a deflection of approximately 30 nm in the BCSR biocantilever, while negative samples lacking COVID RNA showed a deflection of less than 2 nm, a significant difference from the deflection of the positive samples. The results demonstrate that RNA can be detected as a potential target analyte in BCSR sensors. Furthermore, the results demonstrate that removing RNA from an RNA-binding BCSR sensor is effective in "resetting" the BCSR sensor. Legend in the graph: Complement = 100% complementary target, Non-complement = target substance with 80% homology, COVID RNA = purified RNA from the target virus, COVID virus = target COVID SARS-CoV-2 virus, Sputum = human sputum analog without the target substance, Non-COVID = non-COVID-19 coronavirus. The deflection of the baseline or physically dissociated BCSR cantilever was consistently about 1 nm.

[0167] To further evaluate the reliability and reproducibility of the BCSR component assemblies, three different cantilevers (black bar, gray bar, and hash bar) with DNA receptor 008 were tested for detection of four types of analytes (accurate nucleic acid receptor complement, non-complementary nucleic acid, genomic COVID19 RNA complement, and non-COVID coronavirus genomic RNA). The results shown in Figure 11 indicate that all three BCSR sensors produced nearly identical cantilever deflection. Each cantilever had slightly different spring constants, but the positive and negative detection results were consistent. The results demonstrate that BCRS biocantilevers can be reliably and consistently manufactured robustly.

[0168] Figure 12 is a table showing the correlation between receptor-specific curvature and the magnitude of the BCSR sensor response. Three DNA receptors (008, 012c, and 012s) were designed and tested against complementary DNA for hybridization. The difference between 012c and 012s is a single nucleotide change. For hybridization of DNA receptor 008 with its complementary DNA, the calculated maximum shortening of the end-to-end distance is 48 nm. Hybridization of receptor 012c to its complement shortens the distance by 62 nm, while hybridization of receptor 012s to its complement shortens the distance by only 27 nm. Three-dimensional projections (XZ and XY viewpoints) show the relative predicted curvature of each receptor-complement pair. In comparison, the cantilever deflections measured for receptors 008, 012c, and 012s were 28 nm, 32 nm, and 17 nm, respectively. In each case, the magnitude of the cantilever deflection directly correlated with the calculated amount of intrinsic molecular curvature at 58 ± 5%. It is important to note that complete hybridization between complementary chains is inhibited by the fact that the crosslinked receptor chains are permanently attached to the cantilever and stage at both ends, respectively.

[0169] Example 14: Detection of SARS-CoV-2 by bending of a biocantilever Tests were conducted to evaluate the ability to detect multiple pathogen targets, including COVID, influenza (H1N1), adenovirus, and human rhinovirus (HRV). Specifically, the following receptors were targeted: COVID, H1N1 2c, H1N1 3a, Adeno_41, Adeno_49, Adeno_52, HRV A_12, HRV B, and HRV C.

[0170] The table in Figure 13 provides details of receptors designed and manufactured to detect influenza A, adenovirus B, and human rhinovirus (HRV). For each target, two to three different candidate receptors were developed. This redundancy allows for either the selection of the receptor with the greatest detection performance, or the possibility of a multi-system where detection is based on responses to multiple markers in the target genome. Numerous biophysical properties were considered, as listed in the table.

[0171] The columns in the table shown in Figure 13 are defined as follows: Receptor “Source”: A viral segment, specific NCBI / NIH database accession number, or consensus region common to the most dominant variant of the target. (Data corresponding to each identified source can be readily found by those skilled in the art at ncbi.nlm.nih.gov on the World Wide Web, which is incorporated herein by reference.) “Candidate”: The name herein for a particular receptor candidate. “C&S”: A relative measure of the curvature and stiffness of the receptor—larger values ​​directly correlate to a higher detection response. “ENDS”: A measure of receptor curvature calculated by the ratio of the actual length of the receptor along the helical axis to the distance from end to end—larger values ​​directly correlate to an increase in curvature. “% Shortening”: A more intuitive measure of receptor curvature calculated by the actual molecular length compared to the distance from end to end—larger values ​​directly correlate to an increase in curvature. % shortening can be a wide range, e.g., from about 15% to about 80%. "ΔG": Gibbs free energy is a measure of the potential for internal folding of the receptor molecule - a higher value correlates with less internal folding (and thus inhibiting detection) of the target genome marker. Tm℃: A calculated value of the melting temperature of the target genome fragment, important for proper denaturation and hybridization to the receptor. "Target homology": Calculated genetic agreement between the receptor and its target genome fragment - higher homology implies higher specificity. "Maximum % homology to non-target": Agreement rate with the nearest genetic variant, neighbor, or other microorganisms that may be found in the sample - a lower value implies higher specificity.

[0172] To determine the effectiveness of the present invention's approach in distinguishing between multiple pathogens that induce cold and flu symptoms, identified targets were selected. Receptors were designed based on the necessary biophysical properties, including high specificity for the desired detection target and low homology to genetic neighbors. The bar graphs provided in Figures 14A–14I show the responses (filled or striped bars) from five piezoresistive cantilever devices, each tested 10 times and reset between each test. The reset responses consistently showed approximately 5 nm ± 1 nm, as expected from the experimental design in all tests. For comparison between graphs, the vertical scale (nanometers) has been adjusted to 100 nm in all cases. Note that the deviation in the individual target biodetection response (i.e., cantilever deflection) is proportional to the receptor-specific curvature, which was calculated and independently verified. For example, the shortening induced by the curvature over the end-to-end distance of the H1N1 2c receptor (Figure 14B) was predicted to be approximately 81 nm. The measured cantilever deflection was approximately 50 nm. Meanwhile, the predicted cantilever deflection due to the HRV-C receptor (Figure 14I) was 53 nm, while the measured deflection was 33 nm. In both cases, the measured cantilever deflection was approximately 60–65% of the predicted shortening of the receptor length. This observation is not surprising given the incomplete formation of the double helix, and consequently, the degree of structural change was smaller than predicted.

[0173] While the degree of deflection may not always reach the predicted level, the deflection is nevertheless reproducibly proportional to the predicted curvature and large enough for reliable detection. Each sample was tested at least 10 times on multiple cantilevers, using each of the receptors. Furthermore, the results were very consistent between tests. In summary, the binary results of the IDC detection technique demonstrated accurate and consistent detection of the virus in the presence of simulated sputum 10 out of 10 times within seconds.

[0174] Example 15: Other Embodiments Item 1. A sensor assembly for detecting a target analyte, comprising: A cantilever formed from a silicon material having a deflection detection element disposed therein, the cantilever being elongated and having a proximal end and a distal end, the cantilever; A support base having a silicon pedestal disposed on a support surface; A first conductive metallization disposed on the lower surface of the cantilever in electrical contact with the deflection detection element; A second conductive metallization disposed on the upper surface of the silicon pedestal, the second conductive metallization being configured to transmit an electrical signal between the deflection detection element and a contact for electrical communication with an external electrical device; A eutectic bond formed between the first and second conductive metallizations and the pedestal, the eutectic bond being configured to fix the proximal end of the cantilever on the pedestal, the pedestal having a pedestal thickness for supporting the lower surface of the distal end of the cantilever with a fixed gap from the support surface; At least one cross-linking receptor having a first end and a second end, the first end being configured for attachment to the lower surface of the distal end of the cantilever, the second end being configured for attachment to the support surface, the cross-linking receptor being configured to change conformation upon interaction with the target analyte and induce deflection of the cantilever; and at least one cross-linking receptor. The deflection detection element generates an output signal indicating the deflection of the cantilever. The sensor assembly.

[0175] 2. The sensor assembly according to item 1, wherein the deflection detection element comprises a plurality of piezoresistors formed within the silicon material of the cantilever or coated on the surface of the cantilever.

[0176] 3. The sensor assembly according to item 2, wherein the plurality of piezoresistors are configured to define a Wheatstone bridge.

[0177] 4. The sensor assembly described in item 1, wherein the eutectic junction includes a gold-silicon junction.

[0178] 5. A sensor assembly as described in item 1, wherein the fixed gap is in the range of 1 to 1,000 nm.

[0179] 6. The sensor assembly according to item 1, wherein a metallized contact area is formed on the lower surface of the distal end of the cantilever and on the support surface, respectively, for mounting the end of at least one bridging receptor.

[0180] 7. The sensor assembly according to item 6, wherein the metallized contact area contains gold, and at least one crosslinking receptor is thiol-modified to facilitate attachment to the metallized contact area.

[0181] 8. A sensor assembly as described in item 1, wherein at least one crosslinking receptor comprises an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repeat of any one of those molecules, a complex of any one of those molecules, a hybridization of any one of those molecules, or any combination thereof.

[0182] 9. The sensor assembly described in item 8, wherein at least one cross-linking receptor comprises nucleic acid, nucleic acid analog, double-stranded DNA (dsDNA), dsDNA analog, single-stranded DNA (ssDNA), ssDNA analog, or peptide nucleic acid.

[0183] 10. The sensor assembly described in item 8, wherein at least one cross-linked receptor is ssDNA.

[0184] 11. The sensor assembly according to item 7, wherein at least one crosslinked receptor is a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

[0185] 12. The sensor assembly described in item 7, wherein at least one crosslinking receptor is 50 to 1,000 nm in length.

[0186] 13. The sensor assembly described in item 7, wherein the cross-linked receptor undergoes a length reduction of approximately 15% to 80% upon interaction with the target analyte.

[0187] 14. A sensor assembly as described in any one of items 1 to 13, wherein the analyte includes an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbon nanotube, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repeat of any one of those molecules, a complex of any one of those molecules, a hybridization of any one of those molecules, or any combination thereof.

[0188] 15. A sensor assembly as described in item 14, wherein the analyte includes complementary nucleic acid, complementary nucleic acid analog, complementary dsDNA, complementary dsDNA analog, complementary ssDNA, complementary ssDNA analog, complementary RNA, or complementary RNA analog.

[0189] 16. The sensor assembly described in item 14, wherein the analyte is ssDNA.

[0190] 17. The sensor assembly described in item 14, wherein the analyte is RNA.

[0191] Item 18. Analyte detection system, A test well or chamber configured to accept one of the sensor assemblies from item 1 to 13, An analyte detection system comprising a sensor assembly and a conductive connector configured for electrical communication between the sensor assembly and an instrument for generating an external display indicating the detected deflection of the cantilever.

[0192] 19. The analyte detection system described in item 18, wherein the test well or chamber is configured to hold a sample containing one or more of the following: tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal specimens, nasopharyngeal specimens, exhaled breath specimens, urine, feces, tissue, blood, plasma, serum, cell cultures, body fluids, tissue biopsies, apocrine or eccrine fluids, forensic specimens, aerosols, soil, water specimens, food, ingredients, raw materials, in-process specimens, by-products, products, or quality control specimens.

[0193] 20. The analyte detection system described in item 19, wherein the sample includes viruses, bacteria, phages, yeasts, mycoplasmas, fungi, human cells, animal cells, plant cells, insect cells, or any combination thereof.

[0194] 21. The analyte detection system described in item 19, wherein the sample is a mucus sample, nasal sample, breath sample, or nasopharyngeal sample.

[0195] 22. An analyte detection system as described in item 19, in which the sample contains a virus.

[0196] 23. An analyte detection system as described in item 18, wherein the virus is one or more of the following: coronavirus, influenza virus, respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or Zika virus.

[0197] The integrated NEMS cantilever sensing devices and approaches disclosed herein provide accurate, reproducible, and cost-effective tools for detecting pathogens and other biomarkers. Sensor units can be manufactured at very low cost, with a size of 100 microns and a weight of less than 1 milligram, using established MEMS manufacturing techniques and technologies. Using the exemplary manufacturing processes described, large-scale, reliable, consistent, and reproducible detection units with high specificity can be manufactured and used for analyte detection without requiring separation and purification processes. The optional, resettable flow cell embodiments offer significant versatility, along with applications in clinical settings such as agriculture, food, and water safety, as well as public health environments (e.g., schools and customs checkpoints), and large-scale sample analysis. Unlike many existing diagnostic tests with limited shelf lives for detection reagents, many elements of the present invention's approach (e.g., MEMS and electronic components) can be stored for extended periods without the risk of degradation. The ability to rapidly sequence and synthesize DNA makes the present invention's approach a practical and cost-effective tool for preparing for detection and containment in future outbreaks of infectious diseases.

Claims

1. A sensor assembly for detecting a target analyte, A cantilever formed from a silicon material having a deflection detection element disposed inside, wherein the cantilever is elongated and has a proximal end and a distal end, A support base having a silicon pedestal placed on the support surface, A first conductive metallization is provided, which is in electrical contact with the deflection detection element and positioned on the lower surface of the cantilever. A second conductive metallization disposed on the upper surface of the silicon pedestal, wherein the second conductive metallization is configured to transmit an electrical signal between the deflection detection element and a contact that communicates with an external electrical device, A eutectic joint formed between the first and second conductive metallizations and the pedestal, wherein the eutectic joint is configured to fix the proximal end of the cantilever onto the pedestal, and the pedestal has a pedestal thickness for supporting the lower surface of the distal end of the cantilever with a fixed gap from the support surface, The present invention comprises at least one bridging receptor having a first end and a second end, wherein the first end is configured for attachment to the lower surface of the distal end of the cantilever, and the second end is configured for attachment to the support surface, and the bridging receptor is configured to change conformation upon interaction with the target analyte, thereby inducing deflection of the cantilever. A sensor assembly in which the deflection detection element generates an output signal indicating the deflection of the cantilever.

2. The sensor assembly according to claim 1, wherein the deflection detection element comprises a plurality of piezoresistive elements formed within the silicon material of the cantilever or coated on the surface of the cantilever.

3. The sensor assembly according to claim 2, wherein the plurality of piezoresistors are configured to define a Wheatstone bridge.

4. The sensor assembly according to claim 1, wherein the eutectic bond includes a gold-silicon bond.

5. The sensor assembly according to claim 1, wherein the fixed gap is in the range of 1 to 1,000 nm.

6. The sensor assembly according to claim 1, wherein a metallized contact area is formed on the lower surface and the support surface of the distal end of the cantilever for mounting the end of the at least one crosslinking receptor.

7. The sensor assembly according to claim 6, wherein the metallized contact region comprises gold, and the at least one crosslinking receptor is thiol-modified to facilitate attachment to the metallized contact region.

8. The sensor assembly according to claim 1, wherein the at least one crosslinking receptor comprises an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repeat of any one of these molecules, a complex of any one of these molecules, a hybridization of any one of these molecules, or any combination thereof.

9. The sensor assembly according to claim 8, wherein the at least one crosslinking receptor comprises a nucleic acid, a nucleic acid analog, double-stranded DNA (dsDNA), a dsDNA analog, single-stranded DNA (ssDNA), an ssDNA analog, or a peptide nucleic acid.

10. The sensor assembly according to claim 8, wherein the at least one crosslinking receptor is ssDNA.

11. The sensor assembly according to claim 7, wherein the at least one crosslinked receptor is a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

12. The sensor assembly according to claim 7, wherein the at least one crosslinking receptor has a length of 50 to 1,000 nm.

13. The sensor assembly according to claim 7, wherein the crosslinked receptor undergoes a length reduction of approximately 15% to approximately 80% upon interaction with the target analyte.

14. The sensor assembly according to any one of claims 1 to 13, wherein the analyte comprises inorganic molecules, organic molecules, polymers, polymer analogs, carbon nanotubes, carbohydrates, carbohydrate analogs, nucleic acids, nucleic acid analogs, proteins, protein analogs, antibodies, antibody analogs, repeats of any one of the molecules thereon, complexes of any one of the molecules thereon, hybridizations of any one of the molecules thereon, or any combination thereof.

15. The sensor assembly according to claim 14, wherein the analyte comprises a complementary nucleic acid, a complementary nucleic acid analog, a complementary dsDNA, a complementary dsDNA analog, a complementary ssDNA, a complementary ssDNA analog, a complementary RNA, or a complementary RNA analog.

16. The sensor assembly according to claim 14, wherein the analyte is ssDNA.

17. The sensor assembly according to claim 14, wherein the analyte is RNA.

18. An analyte detection system, A test well or chamber configured to receive the sensor assembly according to any one of claims 1 to 13, An analyte detection system comprising: a sensor assembly and a conductive connector configured for electrical communication between the sensor assembly and an instrument for generating an external display indicating the detected deflection of the cantilever.

19. The analyte detection system according to claim 18, wherein the test well or chamber is configured to hold a sample comprising one or more of the following: tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal specimens, nasopharyngeal specimens, exhaled breath specimens, urine, feces, tissue, blood, plasma, serum, cell cultures, body fluids, tissue biopsies, apocrine or eccrine solutions, forensic samples, aerosols, soil, water samples, food, components, raw materials, in-process samples, by-products, products, or quality control samples.

20. The analyte detection system according to claim 19, wherein the sample includes viruses, bacteria, phages, yeast, mycoplasma, fungi, human cells, animal cells, plant cells, insect cells, or any combination thereof.

21. The analyte detection system according to claim 19, wherein the sample is a mucus sample, a nasal sample, a breath sample, or a nasopharyngeal sample.

22. The analyte detection system according to claim 19, wherein the sample contains a virus.

23. The analyte detection system according to claim 18, wherein the virus is one or more of the following: coronavirus, influenza virus, respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or Zika virus.

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