Integrated MEMS sensor with bridged conformational shift receptor

Abstract

The invention provides an integrated NEMS cantilever sensor with a precisely controlled cantilever gap, having a receptor attached to bridge the gap. When the receptor interacts with the target analyte, it changes the conformation, thereby causing a change in the gap. A detector within the sensor generates a signal corresponding to a change in gap size to confirm interaction with the analyte.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 488,865, filed on March 7, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This application generally relates to devices having microelectromechanical systems (MEMS) sensors, and more specifically to devices and methods for detecting, identifying and quantifying target analytes. Background Technology

[0004] Existing analyte diagnostic techniques employ basic chemistry and analytical instruments such as spectrophotometry, gas chromatography, or liquid chromatography. In the context of this discussion, an analyte is defined as any inorganic or organic compound or material for which information is sought, including its identity, purity, quantity, and / or concentration. For example, currently employed pathogen sensing techniques utilize enzyme-based immunoassays, polymerase chain reaction (PCR), fluorescence signal transduction, or quartz microbalances and silica microcantilever arms to determine the presence and / or quality of a target pathogen. Unfortunately, existing techniques generally lack the speed, accuracy, and sensitivity to provide end-users with sufficiently informed decision support. The presence of background materials often reduces the accuracy of these techniques and therefore requires extensive sample preparation prior to diagnosis. Furthermore, these techniques are so cumbersome and complex to operate that they are prone to false positives and false negatives.

[0005] Addressing the needs of the modern analytical diagnostics market means developing technologies that are sensitive enough to detect relatively few molecules as quickly and accurately as possible, unaffected by environmental background materials. New materials, nanotechnology, and micro-biomechanical devices have made significant progress towards this goal.

[0006] Microelectromechanical systems (MEMS) have been identified as a promising technology with the potential to revolutionize both industrial and consumer products by combining silicon-based microelectronics with micromachining techniques. MEMS as a manufacturing technology offers several distinct advantages, including the interdisciplinary nature of MEMS technology and its micromachining techniques, and the diversity of its applications, which has led to an unprecedented range of devices and synergies with previously unrelated fields such as biology and microelectronics. Furthermore, MEMS mass production technologies allow for the manufacture of parts and devices with increased performance and reliability in configurations with reduced physical size, volume, weight, and cost. In addition, MEMS provides a foundation for manufacturing products that cannot be manufactured by other methods. These factors make MEMS a potentially superior detection technology.

[0007] Currently, most MEMS applications exist in systems used in automotive, electronics, communications, and defense applications. Their unique mechanical properties and miniaturized size make MEMS sensors particularly suitable for use as accelerometers (airbag sensors), inkjet printer heads, computer disk drive read / write heads, projection display chips, blood pressure sensors, optical switches, and miniature valves.

[0008] Advances in MEMS fabrication technology have greatly facilitated the development of biosensors in medical applications. These advancements include micromachining of bulk surfaces, 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 its mechanical properties and / or both.

[0009] Cantilevers are one of the fundamental components of MEMS, with dimensions ranging from micrometers to nanometers. 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, a solid structure is released from the substrate to create a freestanding beam anchored at one end. Cantilevers can be fabricated as a single unit or an array of units and can be designed with varying degrees of “stiffness” or flexibility to meet the requirements of specific applications. These processes also allow for the fabrication and integration of electronic circuitry and other MEMS components required to interface with the cantilevers. Their flexibility and versatility make them a promising component for a wide range of environmental, biomedical, and consumer applications. Existing biosensing applications typically integrate MEMS cantilevers within a test chamber to isolate the receptor-analyte reaction to a minimum volume.

[0010] In conventional MEMS sensor cantilever arms, bending in both static and dynamic modes is achieved through changes in mass and / or Young's modulus. Unlike gravity in the static mode, the dynamic mode utilizes oscillations as the driving force. However, these conventional MEMS cantilever arms have significant limitations.

[0011] Cantilever sensors can operate in static and / or dynamic modes. In static mode, molecular interactions on the cantilever surface translate into cantilever bending due to variations in surface stress. In static mode, various techniques, such as diffraction or reflection of light or piezoresistive materials, can be used to measure displacement or deflection within the cantilever. In dynamic mode, changes in mass or Young's modulus can be measured by oscillating the cantilever's resonant frequency. Young's modulus can cause negative or positive frequency shifts, depending on the thickness of the analyte deposition layer. Dynamic mode is more sensitive to changes in cantilever properties than static mode. It should be noted that for biological cantilever devices known in the art, sufficient mass must be deposited on the device to influence a detectable response. Furthermore, non-specific deposition from background materials is a known challenge for such biological cantilever devices, requiring cantilever surface masking or sample preparation or purification to achieve sufficient specificity.

[0012] The dynamic pattern can be described by the following mathematical equation.

[0013] The force balance equation for the cantilever is:

[0014] (1)

[0015] Where k is the spring constant and y0 is the neutral axis. To obtain the optimal performance due to mass change, assume no damping (γ=0), a simple linear spring, and that a transient force F has been applied. ext = 0. If Δy = (y0 – y), then equation (1) simplifies to:

[0016] (2)

[0017] Solving the derivative yields , and , where ω0 is the fundamental natural oscillation frequency of the cantilever.

[0018] The resonant frequency of the cantilever before any mass is added is

[0019] (3)

[0020] And after adding quality, for

[0021] (4)

[0022] Where m a = m b + Δm and k a = mk b + Δk.

[0023] By taking the logarithm and then differentiating, equation (3) is modified, resulting in equation (5):

[0024] ,or (5)

[0025] This indicates that the resonant frequency shift is a function of the mass change Δm and the spring constant change Δk.

[0026] If the spring constant remains constant during mass deposition, then the change in frequency is a function of the change in deposition mass, making Equation 5 simplify to

[0027] (6)

[0028] Substituting into the above equation, equation 6 becomes

[0029] (7)

[0030] in and .

[0031] The relevant mass of the cantilever before adding mass is its volume V. b Multiply by the density ρ and the geometric coefficient α1. For a straight beam fixed at one end, α1 = 0.24.

[0032] V b = LWH and m b = α1ρWLH.

[0033] Using these relations, equation (7) becomes

[0034] (8)

[0035] The change in resonant frequency is proportional to the change in mass multiplied by a set of constants that define the characteristics of the cantilever.

[0036] If the change in mass load on the cantilever is negligible compared to the change in the spring constant, then equation (5) simplifies to

[0037] (9)

[0038] in .

[0039] The change in the cantilever's spring constant as a function of the height of the analyte deposited or adsorbed onto the surface is determined by taking the differential of k with respect to H in Equation 9, which provides an approximation for a small ΔH. Additional analyte essentially alters Young's modulus or cantilever stiffness.

[0040] (10)

[0041] Where dH~Ns A t H t N s A represents the number of analyte components on the surface of the cantilever. t The surface area covered by the analyte component, and H t The cantilever constant represents the height of each analyte component. For typical applications reported in the literature, the cantilever constant can vary from 5% to as high as 40%.

[0042] The response of the cantilever to a change in mass relative to a change in Young's modulus can be calculated. The points where the relative frequency changes due to the mass load (negative) and Young's modulus (positive) are equal are also considered. This occurs when there is no significant shift in the resonant frequency due to the addition of analyte components to the cantilever surface, i.e.,

[0043] or The condition is thus satisfied. (11)

[0044] The masses of the cantilever before and after mass addition are respectively

[0045] m b = α1ρ b WLH, and

[0046] m a = α1ρ b WL(H + dH c ), where H c This represents the critical height for the analyte to stratify.

[0047] The spring constant k before adding the additional analyte is obtained from equation (9) above. After adding the analyte, the spring constant becomes...

[0048] (12)

[0049] Make

[0050] (13)

[0051] Where E b E represents the Young's modulus of the cantilever before the addition of the analyte. a ρ represents the Young's modulus of the cantilever after the addition of the analyte component. b The density of the cantilever before adding the analyte component, and ρ a This represents the density of the cantilever after the addition of the analyte. Typical values ​​for cantilever densities can produce dH values ​​on the order of 20 nm to 30 nm. c .

[0052] MEMS cantilever in aqueous solution, i.e., not in air, presents additional challenges for detection due to the significant damping effect on the dynamic motion of the cantilever. The solution to equation 1 becomes:

[0053] (14)

[0054] (15)

[0055] Where Q is the quality factor, and

[0056] (16)

[0057] Where ω' is the damping resonant frequency of the cantilever in aqueous solution. It has a very low damping coefficient in air. Compared to cantilevered surfaces, aqueous solutions also exhibit frequency peak broadening. This allows the detection upper limit to be within the picometer range.

[0058] Static mode detection does not induce oscillations, and therefore does not introduce acceleration or velocity terms into the system's force balance equations. For a typical cantilever spring, this is on the order of several 10,000 Gs at the resonant frequency in the MHz range. In static mode, the force on the cantilever originates from the Earth's gravitational field, where the force is simply F = mg, and g is the acceleration due to gravity.

[0059] Therefore, the force balance equation for the cantilever operating in static mode simplifies to ky = F ext = mg.

[0060] Taking the logarithm and the differential produces

[0061] (17)

[0062] Or for displacement, (18)

[0063] Similar to the dynamic mode, the displacement of the static mode system is a function of both the change in mass and the change in the spring constant.

[0064] For a cantilever spring system with a constant spring constant, the equations (18) can be rearranged based on the displacement caused by the increase in mass, i.e.,

[0065] (19)

[0066] Because the total mass change caused by typical mass loads on a MEMS cantilever is very small, the cantilever displacement or deflection is also very small—on the order of femtometers (fm). The magnitude of this displacement is unsuitable for known transduction methods. In the absence of acceleration and velocity terms contributing to the equations of motion, purely static detection modes are significantly inferior to dynamic detection modes.

[0067] When the relative mass load is small, the effect of the relative change in the spring constant on the displacement is greater than that of the change in the spring constant.

[0068] (20)

[0069] The change in Young's modulus caused by surface material modification is independent of mass load and is difficult to measure. Therefore, small displacements associated with purely static methods are not suitable for detection schemes.

[0070] Nevertheless, the static mode can be improved by employing additional force to effectively reduce the apparent cantilever spring constant. Adding power via a capacitor configuration can significantly improve the response to changes in mass load, but modifying the effective spring constant has its limitations due to the highly nonlinear behavior of the dynamic power that alters the plate distance (cantilever-base clearance).

[0071] The cantilever surface can be further coated or functionalized to enhance performance. To detect specific analytes in a sample, the cantilever surface can be coated or functionalized with layers of specific chemical or biomolecules. Cantilever surface functionalization is typically performed after the sensor is manufactured, as the harsh deposition and etching processes can damage any pre-existing functionalization. Functional layers can be coated on one side of the cantilever surface or on any specific and localized area of ​​the cantilever surface. The quality of the functionalization directly affects the performance of the sensor signal.

[0072] Cantilever surface modification for chemical, biological, and biomedical applications falls into two categories: physical modification and chemical modification. Physical modification alters the surface morphology or shape while leaving the chemical properties largely unchanged, such as etching, sandblasting, and machining. Established chemical techniques include plasma and chemical vapor deposition, atomic layer deposition, and electrochemical deposition. Chemical treatments can result in surface oxidation / nitriding / carburization, surface functionalization, ion implantation, monolayer coatings, or coatings consisting of multiple layers with different compositions. The objectives of modifying cantilever surfaces can include (1) producing specific surface morphologies; (2) improving biocompatibility; (3) altering surface composition; and (4) creating layers of material with specific chemical compositions.

[0073] Numerous tools have been developed to address the challenges of functionalizing cantilever surfaces. Microcapillaries are relatively simple to use and suitable for small-scale functionalization of cantilevers. Micromanipulators with translation stages precisely position chemically-filled microcapillaries to approach cantilever sensors. Capillaries enable certain cantilever sensors to access chemical solutions for functionalization. Another efficient method for functionalizing cantilevers is using chemical inkjet printing, a technique used to fabricate high-density DNA and protein microarrays. Functionalization of self-assembled monolayers, polymer solutions, and DNA samples has demonstrated comparable performance on cantilever sensors. Using this approach, microcantilever sensors can be functionalized in batches at the wafer level. Other possible variations of the cantilever are 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 processing is provided in U.S. Patent No. 7,433,811 to Gao et al., which is incorporated herein by reference.

[0074] A further extension of MEMS as sensors is bio-MEMS, which involves the use of small biomechanical structures that respond to changes in the environment. These changes might include the introduction of organic or inorganic analytes, or thermal changes in the environment. The biological components of Bio-MEMS, called "receptors," respond to these environmental changes, and this response can translate into movement of the mechanical structures.

[0075] Bio-MEMS sensor technologies encompass various device designs, signal generation and transduction modes, and the orientation of biological components. Typically, the top surface of a MEMS cantilever is coupled with receptor molecules that specifically bind to unique target molecules. For example, immobilized antibody receptors will interact with specific antigen targets. Therefore, the receptor-target complex significantly alters the mass of the MEMS device, leading to changes in its orientation, deflection, or resonance. Bio-MEMS can operate in static or dynamic modes. In static mode, the association of the target molecule with the receptor-coated surface induces stress, which causes the cantilever to bend. In dynamic mode, the resonant frequency of the cantilever decreases as the target molecule adsorbs onto the receptor-coated surface. Any detection method requires a large number of receptor-target complex molecules to induce significant changes in the position and / or resonance of the MEMS sensor. These techniques may lack specificity, exhibit very poor sensitivity, and are extremely difficult to manufacture on a large scale continuously.

[0076] Signal transduction or measurement of cantilever deflection can be achieved through interferometry or beam deflection away from the free end of the cantilever, commonly seen in AFM (atomic force microscopy) instruments. This method requires a laser source and a position-sensitive photodetector, which often necessitates frequent calibration and consistent sample refractive index. Therefore, this approach often fails in applications outside of controlled laboratory environments. Another common approach is to apply a piezoresistive material to the cantilever, whereby stress causes changes in conductivity and / or resistance.

[0077] In other prior art, various molecules have been envisioned to bridge the gap between opposing electrodes. For example, US 8,078,408 and US 9,857,366 by Albert et al. (incorporated herein by reference), and US 10,036,064 and US 2019 / 0094175 by Merriman et al. teach bridging electrodes that complete the circuit and / or suppress resonance due to the high A / G composition of conductive DNA. In Merriman's disclosure, the association of the bridging molecule with molecules in its environment does not cause mechanical deflection of the electrode. Albert's '408 and '366 patents describe bridging the gap between the free ends of two surfaces to form a circuit using template molecules of ssDNA. After hybridization with the target molecule, dsDNA increases the conductivity of the entire circuit. Although the written description mentions alternative methods for measuring cantilever deflection using piezoresistive methods, the lack of achievable disclosures, combined with the inventors' subsequent inability to fabricate a functional device with any degree of reproducibility in terms of gap and behavior, led to abandonment of this effort.

[0078] Among the rich diversity of molecular interactions, nucleic acid hybridization occurs when single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal and hybridize with complementary DNA or RNA molecules. Methods and devices for detecting biological targets have applied the fundamental principle of hybridization between complementary nucleic acid base pairs, such as cytosine-guanine bonding and adenine-thymine or uracil bonding. Known methods involve exposing specific nucleotide sequences, typically bound to radioactive, fluorescent, or chromogenic tags, to an array of nucleic acid fragments covalently attached to a surface or material such as nylon or nitrocellulose. Hybridization occurs if the nucleotide sequence of the labeled “probe” is complementary to a fragment within the molecular array bound to the surface, and detection is determined by the presence of a radioactive, fluorescent, or chromogenic signal. Typically, the transduction techniques described above require large amounts of analyte, leading to a reduced limit of detection.

[0079] Sensing methods and devices that utilize intrinsic or inherent molecular structures, as well as changes in molecular structure due to variations in the surrounding environment, are 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 a resident molecule through various interactions, such as molecular repulsion, hydrophilicity, ionic bonding, etc. As an example, a neuroprotein called a prion can transform from an α-helix to a β-sheet conformation in the presence of another β-sheet prion. As another example, introducing a single-stranded DNA molecule into the presence of a complementary DNA strand can lead to hybridization to form a double-stranded DNA helix. It is known in the art that double-stranded DNA molecules exhibit an intrinsic curvature for key biological functions such as nucleosome localization and genetic expression. Various theoretical models have been proposed to explain the relationship between the DNA nucleotide sequence and the deflection of the helical axis, or the intrinsic DNA curvature. These models share commonalities in the number, position, and length of adenine nucleotide bundles (called A bundles). The hydrogen bonds between guanine and cytosine residues are perpendicular to the helical axis. Conversely, the bonds between adenine and thymine residues have a distinct 2° to 4° angle per base pair. Therefore, the A bundle located on the same side of the DNA helix can induce the intrinsic curvature of the molecular axis, thereby significantly reducing the end-to-end distance of the double-stranded nucleotide sequence. Summary of the Invention

[0080] The apparatus and methods disclosed herein relate to the detection of virtually any analyte, regardless of sample type or background material within the sample. In some embodiments, the apparatus includes one or more integrated sensors configured to form a bridging cantilever geometry. The integrated sensor includes a first surface, a second surface (one of which is flexible), and a receptor. The receptor includes 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, when the receptor associates with the analyte, the receptor is in a configuration physically associated with the analyte. In some embodiments, when the receptor does not associate with the analyte, the receptor is in a configuration physically dissociated from the analyte. When the receptor interacts, associates, or dissociates with the analyte, a detector generates an output signal.

[0081] A sample preparation reagent (SPR) and a method for detecting analytes in a sample using the SPR are also provided. The SPR comprises a pH buffer component, an enzyme activity inhibitor, a denaturant, a low-ionic-strength detergent, and an oxidant. The buffer component maintains a pH that supports the dissociation of the analyte from its ligands, native complement, or intrinsic secondary structure. The low-ionic-strength detergent disrupts host viral or cell wall structures to release the analyte from the cell. The oxidant produces controlled oxidative fragmentation of the analyte.

[0082] In one aspect, a sensor assembly for detecting a target analyte includes: a cantilever formed of silicon having a deflection detection element disposed therein, the cantilever being elongated and having a proximal end and a distal end; a support base having a silicon substrate disposed on a support surface; a first conductive metallization layer disposed on a lower surface of the cantilever and in electrical contact with the deflection detection element; a second conductive metallization layer disposed on an upper surface of the silicon substrate, the second conductive metallization layer being configured to conduct an electrical signal between the deflection detection element and a contact electrically connected to an external electrical device; and eutectic bonding formed on the first conductive metallization layer. The eutectic bonding between the metallization layer and the second conductive metallization layer and the base is configured to fix the proximal end of the cantilever to the base, wherein the base has a base thickness to support the lower surface of the distal end of the cantilever at a fixed gap from the support surface; and at least one bridge acceptor having a first end and a second end, wherein the first end is configured to attach to the lower surface of the distal end of the cantilever, and the second end is configured to attach to the support surface, wherein the bridge acceptor is configured to change conformation and cause deflection of the cantilever when interacting with the target analyte; wherein the deflection detection element generates an output signal indicating the deflection of the cantilever.

[0083] In some embodiments, the deflection detection element includes a plurality of varistors formed within the silicon material of the cantilever or coated on the surface of the cantilever. The plurality of varistors may be configured to define a Wheatstone bridge. The eutectic bonding may be gold-silicon bonding. In some embodiments, the fixed gap is in the range of 1 to 1,000 nm. Metallized contact regions may be formed on each of the lower surface and the support surface at the distal end of the cantilever for attaching the end of at least one bridge acceptor. The metallized contact regions may be gold, wherein at least one bridge acceptor is thiol-modified to facilitate attachment to the metallized contact regions.

[0084] At least one bridging acceptor 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 of these molecules, a conjugate of any of these molecules, a hybrid of any of these molecules, or any combination thereof. In some embodiments, at least one bridging acceptor may be a nucleic acid, a nucleic acid analog, double-stranded DNA (dsDNA), a dsDNA analog, a single-stranded DNA (ssDNA), an ssDNA analog, or a peptide nucleic acid. At least one bridging acceptor may be a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The length of at least one bridging acceptor may be from 50 to 1,000 nm, and may further undergo a length reduction from about 15% to about 80% after interaction with the target analyte.

[0085] In some embodiments, the analyte detected by the sensor assembly can be 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 of these molecules, a conjugate of any of these molecules, a hybrid of any of these molecules, or any combination thereof. The analyte can be 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. The analyte can be ssDNA or can be RNA.

[0086] On the other hand, the analyte detection system includes a test well or test chamber configured to receive the sensor assembly described above, and a conductive connector configured for electrical communication between the sensor assembly and the instrument to generate an external display indicating the deflection of a detected cantilever. The test well or test chamber can be configured to retain a sample, which may be one or more of the following: tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal sample, nasopharyngeal sample, respiratory sample, urine, feces, tissue, blood, plasma, serum, cell culture, body fluid, tissue biopsy, apocrine fluid, or exocrine fluid, forensic sample, aerosol, soil, water sample, food, ingredients, raw materials, process sample, by-product, product, or quality control sample. The sample may include viruses, bacteria, bacteriophages, yeast, mycoplasma, fungi, human cells, animal cells, plant cells, insect cells, or any combination thereof. The sample may be a mucus sample, nasal sample, respiratory sample, or nasopharyngeal sample. 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.

[0087] The present invention utilizes the end-to-end length variation of the attached receptor. The change in receptor length corresponds in magnitude to the change in deflection / displacement distance of the MEMS cantilever. This method is independent of the physical properties of the MEMS cantilever, as long as the receptor curvature strength exceeds the force constant of the cantilever. More specifically, length, thickness, width, manufacturing material, surface properties (Young's modulus), and attachment point are not applicable to this method. The advantage of this method is that the manufacturing process, manufacturing material, and physical properties of the MEMS cantilever are not critical to the successful implementation of analyte detection and identification. It should be noted that the attachment point and / or cantilever length will affect the detection sensitivity because these parameters alter the magnitude of the MEMS cantilever's deflection / displacement. The degree of sensitivity variation can be calculated based on the physical properties of the transconducting material (i.e., the piezoresistor). The shorter the cantilever and the closer the receptor attachment is to the clamping point, the higher the sensitivity. However, since the preferred embodiment simulates a binary system with respect to deflection, and the signal is experimentally measurable in this application, sensitivity is not a parameter that needs improvement. Furthermore, it depends on the relative change rather than the absolute value of the transduction value, which alleviates the need to know the absolute initial deflection / displacement position of the MEMS cantilever and the specific value of the piezoresistor on the MEMS cantilever. Each MEMS cantilever with an acceptor can potentially have a variety of initial unresponsive values ​​known before the introduction and challenge of the analyte. Attached Figure Description

[0088] Figure 1 A to 1D schematically illustrate the use of static ( Figure 1 (A to 1B) and dynamic ( Figure 1 Examples of bridging conformational shift receptors in C to 1D modes.

[0089] Figure 2 An exemplary sensor device configuration is illustrated, wherein the number and / or relative orientation of the bridging conformational shift receptors and cantilevers vary.

[0090] Figure 3 A schematic example of a cantilever sensor according to an embodiment of the present invention is provided, wherein Figure A shows the pre-exposure conditions, and Figures B to F show the conformational changes in the cantilever caused by the BCSR pulling, pushing, rotating, torsional, and combined responses, respectively.

[0091] Figure 4 It is a calculated curve of the relative curvature along the genome of an organism, where the indicated peaks correspond to potential receptor candidates.

[0092] Figure 5A The distal end of the cantilever sensor assembly is schematically shown from left to right before exposure to the analyte, after detection, and after reset; Figure 5B This is a diagram showing the device response to analyte detection and reset.

[0093] Figure 6A This is a schematic top view of an exemplary integrated sensor assembly according to an embodiment of the device of the present invention; Figure 6B This is a schematic diagram illustrating an exemplary Wheatstone bridge as used in an embodiment of a sensor; Figure 6C The illustration shows a schematic process flow for manufacturing a cantilever according to an embodiment of the present invention; Figure 6D The illustration shows a schematic process flow for manufacturing a support base and assembling a cantilever and a support base according to an embodiment of the present invention. Figure 6E A schematic diagram of the integrated sensor of the present invention according to an embodiment is provided.

[0094] Figure 7 A schematic diagram of an exemplary detection system for detecting a target analyte according to an embodiment of the system of the present invention is provided.

[0095] Figures 8A to 8B These are, respectively, a side view and a top perspective view of the optional flow cell assembly used in embodiments of the system of the present invention; Figure 8C An exemplary method for exposing the integrated sensor of the present invention to a target analyte is illustrated; Figure 8D This is a schematic diagram of an alternative embodiment for exposing a sensor to a target.

[0096] Figure 9 This is a flowchart illustrating the sequence of generating the cantilever-BCSR combination and using it for pathogen detection.

[0097] Figure 10 This is a bar graph showing the BCSR deflection when the target analyte is detected.

[0098] Figure 11 This is a bar graph illustrating the consistent manufacturability of BCSR sensors.

[0099] Figure 12 It is a table showing the correlation between the receptor's inherent DNA curvature and the magnitude of the cantilever deflection.

[0100] Figure 13 This is a table listing details of the target receptors used in the diagnostic applications of the systems and methods of the present invention.

[0101] Figures 14A to 14I Bar graphs of test results and cantilever deflection data for nine different targets with various forms and background materials are provided. Each graph also includes the calculated structure and predicted versus actual system response. Detailed Implementation

[0102] Abbreviations and Definitions

[0103] For ease of understanding of this invention, the following definitions apply to several terms and abbreviations used herein:

[0104] As used herein, “MEMS” refers to a microelectromechanical system that integrates microscale devices or systems with combined mechanical and electrical components. MEMS typically fabricated using conventional integrated circuit (IC) processing techniques (e.g., photolithography, electron beam, and / or other patterning methods) can range in size from 1 µm to 1000 µm.

[0105] As used in this article, "NEMS" refers to nanoelectromechanical systems, that is, miniaturized MEMS, ranging in size from 1 nm to 1 µm. MEMS consists of mechanical nanostructures, nanosensors, nanoactuators, and microelectronics.

[0106] As used herein, a “cantilever” is a structural element (beam) that extends horizontally and is supported at one end relative to a reference surface or base. MEMS devices are typically made of silicon due to its advantageous material properties; however, 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.

[0107] As used herein, BCSR refers to a bridging conformational shift acceptor, which can attach to bridging gaps between different surfaces of a MEMS and / or NEMS structure (e.g., between a cantilever surface and a reference surface). BCSR contacts are areas on the cantilever or base. BCSR contacts can be made of a variety of materials, including gold, silicon, silica, glass, quartz, polymers, platinum, titanium, tin, aluminum, nickel, copper, and iron. Surface contacts can be functionalized with alcohol, olefin, alkyne, amine, carboxylic acid, aldehyde, ketone, ester, and ether groups. Modifications on surface contacts can differ from the rest of the cantilever surface modification for chemical, biological, and biomedical applications.

[0108] As used herein, a "receptor" (which may appear interchangeably in uppercase or all lowercase) is a substance that changes its conformation in response to changes in its environment. Receptors comprise one or more molecular structures that act as binding sites for one or more analytes. Upon binding to one or more analytes, the receptor changes to a different conformation. A non-limiting 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 of these molecules, conjugates of any of these molecules, hybrids of any of these molecules, or any combination thereof.

[0109] As used herein, a “sensor” is a device that detects or measures information from its surrounding environment and provides an output signal in response to the detected or measured parameter. A non-limiting list of the information that can be detected or measured includes mechanical information (force, pressure, velocity, acceleration, position), thermal information (temperature, entropy, heat, heat flux), chemical information (concentration, composition, reaction rate), radiation information (electromagnetic wave intensity, phase, wavelength, polarization, reflectivity, refractive index, transmittance), magnetic field information (magnetic field strength, flux density, magnetic moment, permeability), and electrical information (voltage, current, charge, resistance, capacitance, polarization).

[0110] As used in this article, an "actuator" is a device that converts signals into actions. It can generate a force to manipulate itself, other mechanical devices, or the surrounding environment to perform some useful function.

[0111] As used herein, a “transducer” is a device that converts one form of signal or energy into another. Therefore, the term transducer can be used to include both sensors and actuators.

[0112] As used herein, a "nucleotide" is an organic molecule composed of nucleoside and phosphate ester functional groups. Nucleotides serve as monomeric units of deoxyribonucleic acid (DNA) or ribonucleic acid polymers. There are two types of nucleotides—purines and pyrimidines. Specific purines are adenine (A) and guanine (G). Specific pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA, while U is found in RNA.

[0113] Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. They serve as the monomeric building blocks of peptides and proteins. Secondary, tertiary, and quaternary structures influence the properties, function, and conformational dynamics of proteins.

[0114] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a chain of amino acids in which amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chain can be of any length greater than two amino acids. Unless otherwise stated, the terms “polypeptide,” “protein,” and “peptide” also encompass various forms of modification thereof. Such modifications can be naturally occurring or chemically modified. Examples of modifications include, but are not limited to, glycosylation, phosphorylation, myristylation, palmitoylation, ribosylation, acetylation, etc. Modifications also include intramolecular crosslinking and covalent linkages of various parts, such as lipids, flavins, biotin, polyethylene glycol, or derivatives thereof. Furthermore, modifications may also include protein cyclization, branching of the amino acid chain, and crosslinking of the protein. Additionally, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.

[0115] The terms “protein” or “polypeptide” can also cover “purified” polypeptides that are substantially isolated from other polypeptides that are naturally present in the cells or organisms in which the polypeptide is found (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0116] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Additionally, unless the context clearly indicates otherwise, the use of “or” is intended to include “and / or.”

[0117] The following illustrative examples describe various embodiments and applications of the apparatus and methods of the present invention. These examples are not intended to be limiting. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art upon consideration of the specification or practice of the invention as disclosed herein. The specification, together with the examples, is intended to be considered exemplary only, and the scope and spirit of the invention are indicated by the appended claims.

[0118] Example 1: BCSR cantilever configuration

[0119] A physical example of the system of this invention consists of two spring elements anchored at one end to a base or reference surface. The first spring is a NEMS cantilever. The second spring is a bridging conformational shift receptor (BCSR), which in this example utilizes the inherent DNA curvature, or “IDC”, of DNA. A bridging conformational shift receptor (BCSR) is defined as any structure that changes size to generate a normal force in any direction (up or down / push or pull) on the cantilever spring. In this embodiment, the dimensional change is orthogonal to the plane of the cantilever beam. The two springs are attached together near the first end of their respective physical structures, for example, at or near the end of the cantilever and at the functionalized attachment end of the BCSR. The second end of the BCSR is attached to a supporting structure, i.e., a base or surface fixed relative to the cantilever spring. Reference Figure 1 From A to 1D, the basic concept is that when the BCSR reacts or interacts with components in its environment, it contracts by bending (coiling) or axially contracting to pull downward on the lower surface of the cantilever. Complementary BCSR pairs are designed or selected to achieve the desired shape change to induce a measurable force on the attached cantilever spring sensor. Figure 1 Figures A and 1C illustrate exemplary implementations characterized by pre-detection conditions, wherein the receptor is not associated with the analyte and the cantilever is in a neutral position, such as "relaxed". Figure 1 B and 1D show the results of BCSR association with the analyte, where BCSR contractes to pull the distal end of the cantilever toward the base.

[0120] Changes in the conformational shape of the cantilever can be detected using various techniques, including electrical (piezoresistive, inductive, capacitive, continuity (normally open or normally closed configuration) and optical (laser deflection position), quantum tunneling, fluorescence, or other emission, etc.). In an exemplary embodiment, the piezoresistive transduction and BCSR of the cantilever displacement / bending / deflection produce a conformational change orthogonal to the plane of the cantilever beam after undergoing a reaction with complementary components (corresponding to spring contraction).

[0121] The BCSR can also be extended to allow displacement from the neutral cantilever position. Furthermore, the NEMS cantilever can initially be relaxed or broken or altered by shifting, bending, or deflecting BCSR bases paired with its complementary nucleotide sequence. In this case, the cantilever will return to its undisturbed (neutral) position, resulting in a net change in the displacement, binding, or deflection of the NEMS cantilever.

[0122] The displacement of the cantilever can be induced by many different NEMS operating modes. In the "Static Mode – Pre-Reaction System," the cantilever does not exhibit significant displacement exceeding that caused by the combined mass of the cantilever and the BCSR (i.e., the total mass generated by the gravitational field). In the "Static Mode – Post-Reaction System," the displacement arises from the small restoring force contribution of the cantilever assembly, where the displacement is primarily due to conformational shape changes of the BCSR. In the "Dynamic Mode," the BCSR is modeled with an associated force constant k. BCSR In contrast, in previous systems, the receptor spring had high stiffness and held the cantilever in a permanent displacement or deflection position. Using these different operating modes, the stiffness of the BCSR and associated forces can be dynamically assessed according to the method of the invention, wherein the BCSR acts as the focus of activity and imparts high sensitivity, inducing conformational changes in response to interaction, association, and / or dissociation with one or more analytes.

[0123] From a physical and mathematical perspective, the system can be described as having multiple springs connected in series. In an exemplary embodiment, the device is implemented using two springs. The BCSR applies a force to the connected force-sensing cantilever spring after reacting with its complementary pair. The degree of reaction can produce information about the interaction between the relative pairs. However, for a preferred embodiment, if the complementary pair has the necessary degree of nucleotide base pair homology, the DNA will exhibit a significant shape change and thus an associated force on the cantilever. This characteristic produces a binary detection scheme, i.e., if the resulting complementary pair is a significant match or interaction to produce a conformational change in size, it will apply a force, while if it is not a significant match or interaction, little force is observed, and no conformational change is observed. This is an attractive feature that ensures high specificity. Additionally, a bridging conformational shift receptor has sufficient force to affect the displacement of the sensing cantilever spring to be measured. This characteristic provides an extremely high degree of sensitivity.

[0124] Example 2: Static Mode Operation

[0125] For the static mode cantilever in which the cantilever and the BCSR "spring" are connected in series, the force balance equation of the system is:

[0126] (twenty one)

[0127] In a static system, the acceleration and velocity terms are zero, and the equations simplify to:

[0128] k eff (y0 – y) = F ext ,(twenty two)

[0129] Or where Δy = y0 – y,k eff Δy = F ext (twenty three)

[0130] For a system with two springs in series, the effective spring constant is the sum of the two springs, where the effective spring constant is...

[0131] k eff = k c + k BCSSR .(twenty four)

[0132] For a more general system description with n springs (a cantilever with n BCSRs), the effective spring constant would be:

[0133] (25)

[0134] External force F ext for

[0135] Fext = F c + F BCSR (26)

[0136] Equation (23) then becomes

[0137] k eff Δy = F c + F BCSR (27)

[0138] The force applied by the cantilever is F. c = k c Δy, and the force exerted by the bridging conformational shift receptor is F. BCSR =k BCSR Δy, where K BCSR It is the force constant of the bridging conformation shape shift receptor.

[0139] In a series spring system, the forces from the two springs are different. Since the two springs are connected at a common point at the far end of the NEMS cantilever, the displacements on the two springs must be equal, i.e.,

[0140] Δy c = Δy BCSSR (28)

[0141] The shift of the BCSR can now be assessed before and after the reaction with the complementary pair.

[0142] It is important to note that equation (28), which serves as the master equation, is mathematically proven to be independent of the properties of the NEMS cantilever, namely, the spring constant, length, width, thickness, Young's modulus, and / or mass load. It depends only on the dimensional changes of the BCSR, which cause equal changes in the displacement / deflection / bending of the NEMS cantilever.

[0143] Static mode – the system before the reaction

[0144] Before reacting with complementary and attached BCSR (ssDNA or RNA), the boundary conditions are set by adjusting the gap between the cantilever tip and the fixed base to make F BCSRb = 0. At this stage, both ends of the unreacted BCSR are attached and adjusted, so there is no relaxation or only stretching sufficient to cause a slight displacement. The slight displacement will provide positional control to ensure the attachment of the unreacted BCSR. Therefore, the system is in equilibrium F before the BCSR reacts. Tb = F c The displacement of the cantilever spring is due to the mass of the cantilever and the mass of the BCSR, or due to the total mass of the gravitational field.

[0145] m T = mc + m BCSSR (29)

[0146] The total mass of the NEMS cantilever with the attached BCSR is essentially zero m. T ≈ 0. The approximate mass of a typical NEMS cantilever is tens of femts (approximately 10). -14 The mass is on the order of g. A typical large BCSR (e.g., DNA) has an approximate mass of tens of zeptograms (about 10 g). -20 The magnitude is on the order of g. Furthermore, the cantilever spring constant is much larger than the gravitational constant. Therefore, the cantilever does not exhibit significant displacement.

[0147] (30)

[0148] Therefore, before the reaction of BCSR, the displacement of the NEMS cantilever is Δy. BCSSRb = Δy cb ≈ 0, meaning there is no displacement before the reaction with the complementary pair of BCSR. If any measurable displacement exists, it can be recorded as a baseline representing the location of the initial displacement.

[0149] Static mode – post-reaction system

[0150] After introducing the complementary pair, the cantilever spring measures the amount of apparent or effective displacement. If the force of the BCSR is larger than the restoring force of the cantilever, then the displacement of the cantilever in any direction is due to the displacement of the BCSR. Therefore, in equilibrium, Δy c = Δy BCSRa The cantilever measures the apparent or effective displacement, where the total applied force at the connection point at the distal end of the NEMS cantilever is F. ext = F BCSRa + F c .

[0151] if Then the total force is equal to F. ext = F BCSRa The displacement under these conditions has a very small restoring force contribution from the cantilever component, and the displacement is mainly due to the displacement caused by the confirmed shape change of the BCSR.

[0152] (31)

[0153] Some embodiments utilize NEMS cantilever beams defined with spatial coordinates in Euclidean space. In such systems, cantilever displacement is defined as orthogonal to the cantilever beam in the y-direction. The gap between the attachment point on the cantilever and the BCSR attachment base is on the order of nanometers. Cantilever dimensions include 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 Young's modulus E, moment of inertia I, and a force F exerted by the bridging conformational displacement receptor. BCSR .

[0154] (32)

[0155] Wherein the moment of inertia (33)

[0156] Substituting equation (33) into the cantilever displacement equation (32) and rearranging them, we obtain the force constant k. c .

[0157] (34)

[0158] Therefore, the desired cantilever stiffness can be obtained by selecting the cantilever dimensions (L, H, and W) and the manufacturing material (E).

[0159] The BCSR can be attached at different points along the cantilever. The BCSR attached to the distal tip provides the maximum displacement. Since the BCSR attached to the tip is compressed to its maximum value, further compression of the cantilever can also be achieved through other BCSRs. Complementary configurations of the BCSRs, i.e., their compressed forms, are achieved through their attachment to the cantilever. The details of this attachment are discussed as specific embodiments to provide clarity; however, it is not intended to limit it to the specific discussion below.

[0160] It should be noted that the geometric design features and manufacturing materials of the cantilever play a minor role in the cantilever's displacement. Instead, the key factor is the BCSR, which determines the displacement vector along the line between the two attachment points. This vector does not need to be precisely orthogonal to the cantilever beam (i.e., 90°); however, a non-orthogonal configuration can be expected to result in a reduced displacement. If the BCSR reacts... a The structure (i.e., the "spring") is rigid and relatively incompressible, and if multiple different BCSRs with different attachment geometries are combined on the cantilever, the displacement will not exceed the length of the most orthogonally attached BCSR. In the case where multiple BCSRs will be attached to the cantilever, the preferred physical attachment at the tip can be achieved by physically displacing the tip toward the base attachment plate, such that the shortest distance in the gap will orthogonally align the BCSR at the cantilever tip for bonding / attaching. a Due to BCSR a Physical length limitations, BCSR aIt cannot be attached to other areas along the length of the cantilever. (Attaching BCSR) a At the same time, ensure a specific and optimal connection geometry (orthogonal) and attachment location (cantilever tip). When attaching the BCSR... a Subsequently, the complementary pair is either released or restored to an unresponsive state to achieve BCSR. b Different conformations of the system. The system is then prepared for use in the formation of BCSR via its complementary pair. a Conduct testing.

[0161] The NEMS cantilever can be calibrated for specific signal transduction methods. In some embodiments, a piezoresistive transducer is used. The resistive circuit employs a Wheatstone bridge configuration, where all resistors can be placed directly on the NEMS cantilever, providing the advantage of self-calibration against thermal drift, ensuring all resistors are very close together and thus in thermal equilibrium. The potential across the Wheatstone bridge is calibrated for specific resistor values ​​and the piezoresistive response as a function of cantilever displacement to determine the response slope.

[0162] , or (35)

[0163] (36)

[0164] 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 required to confirm a match in the complementary pair (analyte). Once the potential is detected, a significant match or interaction can be confirmed.

[0165] Example 3: Dynamic Mode Operation

[0166] A dynamic mode mathematical model can be derived for the spring attached to the NEMS cantilever, where the BCSR is attached to a fixed structure. Here, it is assumed that the BCSR is flexible to some extent and can be modeled as having an associated force constant k. BCSSR The spring. In static mode operation, the receptor spring is extremely rigid 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 the system are as follows:

[0167] (37)

[0168] in

[0169] (38)

[0170] k eq Substituting into equation (37), we get

[0171] (39)

[0172] Solve equation (39) to determine the resonant frequency, and obtain

[0173]

[0174] or

[0175] (40)

[0176] 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 displacement receptor can be calculated (Equation (40)).

[0177] Example 4: Cantilever Configuration

[0178] Different embodiments of the cantilever configuration can be formed using one or a combination of integrated sensors, wherein the integrated sensors are configured to form a bridging cantilever geometry. As used herein, "integrated sensor" refers to a cantilever having means for measuring the deflection of the cantilever relative to a support base having a fixed gap bridged by a receptor. An exemplary configuration of a device for detecting an analyte in a sample is schematically illustrated in [illustration missing]. Figure 1 A through 1D. Note that although a single cantilever is shown, multiple cantilever arrays can be arranged. The device includes one or more integrated sensors and a detector. Figure 1 Figures A and 1C each illustrate a neutral or physically analyte-dissociated configuration when the receptor does not associate with the analyte. The integrated sensor 2 is configured to form a bridging cantilever geometry including a cantilever 10 having a first surface 12, a second surface 14 on a base 16 (or other stable surface), and a receptor 20 having receptor recognition sites 22. Each of the first surface 12 and the second surface 14 includes one or more contact points for attaching one or more receptors 20. Figure 1 B and Figure 1 D is illustrated in the diagram. Figure 1 A and Figure 1 The apparatus of C illustrates the physical configuration of analyte association when the receptor associates with an analyte. A first end of the receptor is attached to a contact point on a first surface, and a second end of the receptor is attached to a contact point on a second surface. One or more analytes associate with the receptor through one or more molecular structures on the receptor. Due to conformational changes in the receptor in the presence of one or more bound analytes, the first and second ends of the receptor have changed from their resting state. The relative distance and angle between the first and second ends of the receptor differ in the cases of analyte association and non-association.

[0179] Example 5: Other BCSR cantilever configurations

[0180] Different sensor configurations that are neutral or physically dissociated from the analyte Figure 2 Figure AE shows an example where no receptor is associated with the analyte. These non-limiting embodiments illustrate various possible combinations of the BCSR and the cantilever. Figure A shows a single bridging receptor between a fixed surface and a parallel cantilever. Figure B illustrates a possible implementation of multiple bridging receptors between a surface and a parallel cantilever. Figure C shows an exemplary arrangement with a single bridging receptor between a surface and two parallel cantilevers. Figure D shows an exemplary device with a single bridging receptor between two fixed surfaces, whereby analyte association induces stress in the suspended “cantilever” material. Figure E illustrates a sensor configuration with a single bridging receptor between a surface and a cantilever in a planar orientation. Based on the disclosure herein, those skilled in the art will be able to design various different cantilever-receptor combinations to provide sensors.

[0181] Example 6: BCSR receptor

[0182] Polynucleotides, peptides, and polysaccharides can all change their conformation in response to changes in their environment, such as binding to substrates or analytes, altering salt concentration or pH, or phosphorylation. Besides these biopolymers, other materials are known to change their conformation in response to changes in their environment.

[0183] 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 of these molecules, conjugates of any of these molecules, hybrids of any of these molecules, or any combination thereof.

[0184] Receptors comprise one or more molecular structures that act as binding sites for one or more analytes. When binding to one or more analytes, receptors can change conformation by contracting, bending, or otherwise modifying the gap bridged by the BCSR.

[0185] The genetic material of an organism contains far more information than simple nucleotide coding sequences. The patterned organization of specific nucleotides induces higher-order conformations or curvatures in the genetic material. These structures perform important biological functions, such as controlling gene expression, chromosome packaging, transcription initiation and termination, recombination, DNA replication, and nucleosome localization.

[0186] A known example is the variation of the DNA double helix structure, which has an inherent curvature. While single-stranded DNA (ssDNA) does not exhibit an inherent curvature, the hydrogen bonds between adenine and thymine nucleobases are not perpendicular to the helical axis, allowing an inherent deflection of the axis to be induced when binding to certain analytes by altering the composition, position, and length of certain nucleotide sequences. For example, the end-to-end distance of an 85 nm double-stranded DNA (dsDNA) helix can be reduced to 65 nm or greater.

[0187] Figure 4 Sample maps of the sequence-dependent spatial trajectory and intrinsic curvature distribution of the DNA double helix along the DNA molecule are provided. Typically, the nearest-neighbor wedge model is implemented to calculate the overall DNA structure using the following local helical parameters: dinucleotide twist angle, tilt angle, and roll angle. Figure 4 The two peak curvature regions along the adenovirus genome at approximately 4,600 and 26,000 are shown, indicated by arrows.

[0188] In some embodiments, the receptor includes nucleic acid, nucleic acid analog, double-stranded DNA (dsDNA), dsDNA analog, single-stranded DNA (ssDNA), ssDNA analog, or peptide nucleic acid.

[0189] 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.

[0190] In some embodiments, the receptor length is 50 to 1,000 nm. Those skilled in the art will be able to design / generate receptor lengths to optimize performance for specific applications.

[0191] Another known example of conformational variation is the two conformational isoforms of prion proteins. The cellular isoform of prion proteins is the α-helix-rich prion protein (PrPC), while the prion isoform is the β-structure-rich insoluble conformational isoform (PrPSc).

[0192] Example 7: BCSR's response to the analyte

[0193] The analyte can bind to the receptor on the cantilever and trigger a conformational change in the receptor. The analyte and receptor are bound together through non-covalent interactions, including electrostatic, van der Waals, π-effects, hydrophobic, and hydrophilic interactions.

[0194] In some embodiments, the analyte includes 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 of these molecules, conjugates of any of these molecules, hybrids of any of these molecules, or any combination thereof.

[0195] In other embodiments, the analytes include complementary nucleic acids, complementary nucleic acid analogs, complementary dsDNA, complementary dsDNA analogs, complementary ssDNA, complementary ssDNA analogs, complementary RNA, or complementary RNA analogs.

[0196] The methods and apparatus described herein are not limited to any particular sample type. Examples of samples that can be tested using the apparatus and methods of this invention include tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal samples, nasopharyngeal fluid, urine, feces, tissue, blood, plasma, serum, cell cultures, body fluids, tissue biopsies, apocrine fluid, or exocrine secretions. Sample types and sources may also include forensic, aerosol, soil, water, food, ingredients, raw materials, process, by-products, final products, or quality control samples.

[0197] In some embodiments, the sample may be a virus, bacteria, bacteriophage, yeast, mycoplasma, fungus, human cell, animal cell, plant cell, insect cell, or any combination thereof. The method of the present invention has no molecular limitation in its ability to detect any virus (bacteria, fungi, etc.), regardless of nucleic acid, protein, or other composition.

[0198] The association of BCSR with the analyte can induce various conformational changes, thereby causing changes in the relative positions of the cantilever and the reference. Figure 3 Figure AF schematically illustrates the relative motion of an exemplary sensor having a single bridging conformational shift receptor between a surface and a parallel cantilever. Figure 3 Figure A shows an exemplary sensor having a single bridging conformational shift receptor in a physically dissociated (i.e., neutral) configuration from the analyte. Figure 3 Figure B shows an exemplary sensor with a single bridging conformational shift receptor in a configuration physically associated with the analyte, illustrating the pulling effect on the cantilever. Figure 3 Figure C shows an exemplary sensor with a single bridging conformational shift receptor in a configuration physically associated with the analyte, illustrating the pushing effect on the cantilever. Figure 3 Figure D shows an exemplary sensor with a single bridging conformational shift receptor in a configuration physically associated with the analyte, illustrating the rotational action on the cantilever. Figure 3Figure E shows an exemplary sensor with a single bridging conformational shift receptor in a configuration physically associated with the analyte, illustrating the torsional effect on the cantilever. Figure 3 Figure F illustrates an exemplary sensor with a single bridging conformational shift receptor in a configuration physically associated with the analyte, demonstrating the pulling and torsional effects of the cantilever combination. Besides the illustrated example, a wide variety of arrangements are possible. To provide another alternative configuration, Figure 2 The planar orientation shown in Figure E can be modified so that the BCSR causes relative pulling, pushing, rotation, and / or torsion between the cantilever and the supporting surface. Those skilled in the art will be able to modify the receptor configuration for a particular application without excessive experimentation.

[0199] In most embodiments, the interaction, association, or dissociation of the receptor with one or more analytes causes a change in the relative position of the first surface (i.e., the base or reference surface) 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.

[0200] Example 8: BCSR can be reset

[0201] refer to Figure 5A The association of BCSR 52 with the target analyte 54 can cause the inherent curvature or axial contraction of the bridging acceptor 52, the deflection of the piezoresistive cantilever 50 (shown in step 2), and thus a change in resistance. In some embodiments, the device is used once and then discarded after step 2. Other embodiments can be reset by any means (including thermal, electrical, electrochemical, or chemical methods) that separates the acceptor 52 from the analyte 54. The analyte can be destroyed, destroyed, or removed by flushing or emptying the test chamber. In some embodiments, a relatively alkaline buffer solution is flushed through the test chamber to denature the dinucleotide complex and separate the analyte from the acceptor, for example, in step 3. The dissociation of the analyte from the BCSR causes the acceptor to return to its original conformation, relaxes the piezoresistive cantilever, and thus causes a change in resistance, such as Figure 5B As shown.

[0202] Example 9: Signal Transduction

[0203] The function of piezoresistive materials is well known in the art. In short, in piezoresistive materials, changes in physical properties are converted into changes in electrophysical properties. In the case of the cantilever sensor of the present invention, a flexible piezoresistive material is bonded or coated onto a substrate surface, the substrate surface itself being configured to deform when a force is applied to the material. When the substrate surface is bent, the crystal structure of the piezoresistive material also undergoes deformation or stress, thereby changing its resistive characteristics. The sensitivity coefficient is determined as the ratio of the stress change to the resistance change (i.e., pressure / resistance). It is important to note that the transducing material comprises a piezoresistive coating on the substrate surface, not the substrate surface itself.

[0204] The deflection / displacement of the first and / or second surfaces can be measured by placing the transducer element in direct contact with the first or second surface. The signal transduction via the transducer measures changes in physical properties because it relates to changes in electrophysical properties (V, I, R), electromagnetic amplitude, EM field strength, EM frequency, EM position vector, etc. The amplitude of the transduced signal is proportional to the sensitivity coefficient (slope). In some embodiments, a thin coating of flexible piezoresistive material is bonded to the cantilever surface. When the BCSR bends the cantilever surface, it simultaneously bends / deforms the crystal structure of the piezoresistive material. The deformation or stress of the crystal structure alters the resistive characteristics. The sensitivity coefficient is the change in stress divided by the change in resistance (which is the slope: δdisplacement / δresistance or δD / δR).

[0205] The transconducting material is a patterned varistor coating on the surface. Mathematically, Δdisplacement = (δD / δR)Δresistance, or similarly, Δresistance = (δR / δD)Δdisplacement.

[0206] refer to Figure 6A A MEMS cantilever-based sensor assembly 60 is schematically illustrated. The cantilever 62 is formed of silicon or silicon-on-insulator (SOI) coated with a piezoresistive material to generate a detectable electrical signal when force is applied to the arm. In some embodiments, the lower surface of the cantilever may be coated with gold to facilitate bio-attachment with the BCSR. The proximal end of the arm 62 is bonded to a support base 64 having patterned electronic circuitry. It should be noted that the illustrated interconnect patterns are representative only and do not depict the layout of electrical connections in the device. (See Example 7 below, which describes a Wheatstone bridge circuit for measuring resistance changes.) Electrical conductors 66 extend at least partially along the length of the cantilever 62, embedded or otherwise connected to the piezoresistive material within the cantilever to transmit signals generated within the piezoresistive material to various conductors and bonding pads 68 on the support base 64 to provide electrical signals (e.g., V) to external devices. in V out V ccThe base portion 64 is typically bonded to a printed circuit board (not shown) using conventional wire bonding techniques for forming connections. The base portion 64 and the PCB may be encapsulated within an encapsulation 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 attached to the underside of the distal end of the cantilever 62 and the upper surface of the support base 64 to bridge the gap.

[0207] In some embodiments, the MEMS varistor cantilever assembly is manufactured using 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 vapor deposition, sputtering, etc.

[0208] Figure 6C An exemplary process sequence for fabricating a cantilever on a silicon-on-insulator (SOI) wafer according to an embodiment is schematically illustrated. Based on this exemplary sequence, it will be apparent to those skilled in the art that the process sequence can be modified without departing from the general principles of the method of the invention, which is to provide precision cantilevers with repeatable dimensions and performance.

[0209] The starting substrate is a silicon-on-insulator (SOI) wafer, i.e., silicon and oxide (SiO2) on a processing layer. Exemplary starting wafer layer thicknesses can be on the order of 10 μm, 1 μm, and 400 μm, respectively. The steps for fabricating the cantilever structure according to an embodiment of the method of the present invention are as follows: Figure 6C The steps are labeled (a) to (m). Each step utilizes conventional semiconductor processing techniques known in the art, and therefore will not be described in detail.

[0210] (a) Growth of thermal oxide (SiO2) (approximately 500 nm) on a wafer.

[0211] (b) After the structure is patterned, the oxide is etched to expose the processing layer.

[0212] (c) N-type doping and diffusion in the exposed structure.

[0213] (d) Remove surface oxides.

[0214] (e) Thermal oxidation (approximately 100 nm).

[0215] (f) Patterning and oxide etching are performed to open the structure to define the varistor in the Si layer, followed by P-type doping / diffusion. (Ion implantation can also be used for this step.) An exemplary geometry of the varistor is approximately 10 μm wide x 50 μm long (2 x series), with a diffusion depth of approximately 0.25 to 0.5 μm, depending on the doping concentration estimate.

[0216] (g) Oxide etching and growth of thermal oxide (approximately 100 nm).

[0217] (h) Pattern and etch oxide to expose contact windows.

[0218] (i) Deposit a contact metallization layer (Al / 1%Si) and etch it to pattern it.

[0219] (j) A Si3N4 (“SiN”) passivation layer was deposited, patterned, and etched using plasma-enhanced chemical vapor deposition (PECVD).

[0220] (k) Pattern gold or Ti / Au onto the cantilever tip for BCSR attachment.

[0221] (l) Etch the cantilever and cut the channel (see steps (s) and (t) below).

[0222] (m) Etch the wafer to release the device (see step (u) below).

[0223] The resulting device corresponds to a cantilever, i.e., one end of the BCSR will be attached to one of two surfaces. Exemplary dimensions of some embodiments of the cantilever will be on the order of 400 to 450 μm in length, 100 to 115 μm in width, and a thickness on the order of approximately 3.8 to 5 μm. The resonance of the cantilever will be on the order of 15 to 40 kHz. The cantilever will be permanently attached to a raised base orthogonally attached to a rigid surface, i.e., a mounting base, which forms the second component of the sensor device. The fabrication of the mounting base is described below.

[0224] Figure 6D The diagram schematically illustrates a second component (i.e., a mounting base) for manufacturing the sensor and the process flow for assembling the cantilever structure to the base. The base provides electrical connection between the varistor element on the cantilever structure and external interconnects. The steps for manufacturing the base structure according to an embodiment of the method of the present invention are as follows: Figure 6D The elements are labeled (n) to (u). The following steps utilize conventional semiconductor processing techniques known in the art, and therefore will not be described in detail here.

[0225] (n) Starting from the SOI wafer, a thermal oxide layer is grown on the Si surface.

[0226] (o) Polysilicon is deposited (e.g., PECVD) on an oxide surface and patterned to define a base for supporting the cantilever.

[0227] (p) Pattern and etch oxide to form an opening for connection to the substrate ground.

[0228] (q)Ti / Au is deposited on top of the wafer and patterned to define the electrical contacts and gold (or other material) areas for BCSR attachment.

[0229] (r) The upper surface of the cantilever on the processing wafer is positioned relative to the substrate wafer such that the proximal end of the cantilever is aligned with the polysilicon substrate, and the gold region on the distal end of the cantilever is aligned with the gold region on the substrate. (Alignment guides will be patterned on both wafers according to standard semiconductor processing techniques.) Once aligned, the assembly undergoes thermal processing to form a gold-silicon eutectic. As is known, Au-Si eutectic formation occurs at approximately 360°C to 370°C. Exemplary temperatures used to form bonding will be on the order of 410°C to 470°C.

[0230] (s) Etching cantilever and cutting path.

[0231] (t) Carrier attachment and wafer thinning to release the processing layer from the cantilever.

[0232] (u) The device that has finally completed its release.

[0233] The bonding pad formed in step (q) is connected to the corresponding connector on the PCB to provide external connectivity.

[0234] The key steps in this process include steps (o), (q), and (r), the combination of which allows for the formation of a repeatable and well-defined gap between the bottom surface of the cantilever and the upper surface of the base. Specifically, in the contact area near the distal end of the cantilever (in... Figure 6D The gap between the gold contact area (marked as "Gold Contact #1") and the gold contact area directly below the distal end of the cantilever (marked as "Gold Contact #2") is a key structural component of the sensor device of this invention. These gold contact surfaces can be approximately 0.1 nm thick. 2 (For example, the approximate diameter of a single receptor molecule) up to 1,000 nm 2Or on a larger scale. The resulting structure defines a gold-plated / coated gap between two surfaces, which serves as the attachment point for the molecular portion attachment ends. As previously mentioned, although gold is used as the material for the BCSR contact points in the described example, the contact area material can be a variety of other materials, including silicon, silica, 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 capable of bending or deflecting. This physical configuration is used as a NEMS device for the detection unit. The height of the polycrystalline silicon substrate formed in step (o) can be controlled to tight tolerances within a few nanometers using known deposition techniques. The selected substrate thickness, combined with the thickness of the gold contacts (step (q)) and the eutectic bond formed in step (r), which can be expected to be very thin (on the order of 200 Å or less), defines the gap. The gap can vary from 5 nm to hundreds of micrometers, primarily by controlling the thickness defined in steps (o), (q), and (r). This integrated cantilever structure provides a basis for further modification and is suitable for attaching / bonding single or multiple molecular parts to bridge gaps.

[0235] Figure 6E Provided according to Figure 6C and 6D The diagram illustrates a process flow for manufacturing an integrated sensor 100 according to an embodiment of the present invention. (It should be noted that this diagram is not drawn to scale. Instead, dimensions are highly exaggerated to illustrate key components of the integrated sensor of the present invention, including the placement of the receptor in the gap.) The illustration provides key information about the materials used to manufacture the sensor 100.

[0236] The method of the present invention provides the ability to accurately and repeatably control the gap size, overcoming the shortcomings of previous efforts to manufacture reliable cantilever sensing devices for bridging sensing applications.

[0237] In some embodiments, gold thiochemistry is used to attach or chemically bond the ends of the receptor, including ssDNA, to two gold contact regions. Other attachment chemistry is available depending on the molecular structure of the bridge portion. This chemical has been shown to possess the necessary physical strength to maintain attachment during changes in the molecular structure's shape. The NEMS integrated cantilever unit is designed and fabricated with a desired force constant that is relatively weak compared to the forces exerted by the structure within the molecular portion. This configuration provides the necessary conditions for displacement of the cantilever surface during changes in the shape configuration of the molecular portion (e.g., dsDNA). When a single strand (ssDNA) is attached to the integrated NEMS cantilever at each end between the NEMS cantilever and the fixed surface, it is relatively linear and does not exert too much force to bend or displace the flexible cantilever structure to a normal or neutral position, i.e., a flat position. When highly complementary or matched ssDNA binds / reacts with the attached ssDNA element, it forms a corresponding dsDNA helical structure. dsDNA molecules are specifically selected to have a highly flexible structure via modeling and experimental validation. The newly formed curved structure has a force constant much larger than that of the MEMS cantilever, causing it to bend or deflect. Displacement is measured by an attached varistor placed on or integrated into the surface of the NEMS cantilever. The varistor is positioned on the cantilever at a location that will achieve maximum deformation, i.e., close to the base mount, via a Wheatstone bridge configuration. Deformation of the varistor material causes a change in crystal structure, resulting in a change in resistance, which is measured by the change in potential across the Wheatstone bridge design.

[0238] Example 10: Detection System

[0239] Figure 7 A prototype detection system 70 is schematically illustrated for preliminary testing and evaluation of the inventive method for detecting analytes using integrated sensors. This embodiment operates on the principle of a Wheatstone bridge, which is two simple series-parallel arrangements of resistors connected between voltage supply terminals, wherein the resistors are piezoresistive coatings on cantilever 62, which are converted into analog signals.

[0240] Voltage source 72 provides voltage across the Wheatstone bridge defined by PCB 64 and arm 62. Figure 6BA diagram of a semi-active full Wheatstone bridge that can be employed in some embodiments is provided. Sensor assembly 60 can be inserted into connector socket 63, which provides connection to voltage source 72 and preamplifier 74, which receives the signal from sensor assembly 60 for measuring voltage changes from the Wheatstone bridge. The measured voltage change is converted from analog to digital at A / D converter (ADC) 76. Preamplifier 74 provides a 100-gain amplified signal from the Wheatstone bridge to ADC, which is then input to system computer controller 78, which receives the measurement signal and provides control signals for the operation of system 70, including the control, manipulation, and operation of optional flow cell assembly 80, which will be referenced below. Figures 8A to 8B Further description: The computer controller graphically displays data on the cantilever output voltage as a function of time on the screen. The software was developed using NI-DAQmx (version 21.3). The amplified output signal is 20 nm / V.

[0241] In the flow cell control, which can be provided by a computer controller 78, there are XYZ direction control for a precise multi-axis positioning stage 79, flow parameters, and valves for introducing analytes and / or flushing / rinsing the flow cell, wherein the multi-axis positioning stage is used to position the sensor assembly in the test well or test chamber. The computer controller 78 may also include a memory for storing results and links to external memory and / or communication systems for reporting measurement results.

[0242] A magnified optical imaging system was designed, fabricated, and tested to visually observe the positioning and deflection of the MEMS cantilever above a sliding base. A camera captures images / videos of the magnified cantilever and projects them onto a computer screen and / or transmits the visual information to a computer.

[0243] In the test setup, positioning stage 79 is a pressure-sensitive flexure stage mounted beneath the cantilever to adjust the X, Y, and Z positions of the gold-coated surface from which the receptor bridges to the cantilever tip. Precise positioning of the pressure-sensitive flexure actuator unit control console enables nanometer-scale control of the gap distance between the gold surfaces. To achieve a cost-effective and deflection-sensitive cantilever system, an open-loop pressure-sensitive flexure stage with X, Y, and Z torque components of 267 nm / V was selected. For the full length of the 20 μm travel range (at a maximum of 75 volts), the pressure-sensitive flexure element moves 267 nm for every volt applied to each channel. The pressure-sensitive flexure stage, along with all other dynamically moving parts, is mounted on an 18" x 24" test plate optical bench with vibration-isolated feet. A slide retainer attachment fixture is mounted directly to the pressure-sensitive flexure stage. A gold-coated glass slide is attached to the slide retainer attachment to provide the underlying biological receptor attachment area. To adjust the gap between the cantilever and the base, the pressure-sensitive flexure stage is raised or lowered as needed during various experiments. The pressure-sensitive flexure stage can be manually operated using control knobs on the pressure-sensitive flexure control module, or connected to two ports via a USB cable through computer software and a GUI running on a computer. The cantilever mounting fixture consists of several parts: an elevated mounting base and a magnetic cantilever mounting head for easy disassembly and replacement. The cantilever mounting head allows the MEMS cantilever's PCB to be secured to it using an adjustable screw clamping device. Additionally, a microscope camera is mounted on an optical test plate to visually inspect the alignment and bending of the MEMS cantilever during experimental execution.

[0244] In the experimental setup, the amplifier circuit provides a voltage of 2.048 volts across the Wheatstone bridge. This circuit provides a positive potential difference when the MEMS cantilever deflects upward and a negative potential difference when it bends or is pulled downward. Two of the piezoresistors are placed at the base of the cantilever, while two other piezoresistors are placed near the base of the cantilever on the fixed portion of the MEMS device. Placing two piezoresistors at the bottom of the cantilever allows them to experience changes in physical dimensions, such as expansion (bending upward) and compression (bending downward). This physical change in the piezoresistive material on the cantilever provides a transduction means for detecting the degree of bending in the MEMS cantilever. Connector socket 63 allows for quick replacement of the sensor assembly.

[0245] To provide forward control from the MEMS cantilever to the computer output display, the stage is moved upwards using a vernier control knob (coarsely first, then finely) until the gold-coated glass slide just contacts the tip of the MEMS cantilever. As the tip approaches engagement, the MEMS cantilever voltage output is monitored and adjusted to the baseline offset voltage representing zero deflection. The mounted MEMS cantilever is then deflected upwards by moving the pressure-sensitive deflection stage upwards and measuring the voltage on the computer data output window. Changes in the MEMS cantilever voltage are monitored to check for appropriate deflection changes during upward and downward pressure-sensitive deflection controller scans, i.e., voltage amplitude and voltage direction (+ / -). This process ensures that the MEMS cantilever and pressure-sensitive deflection stage respond in the desired manner before attaching the acceptor and performing the detection experiments.

[0246] Preliminary testing involved bending the cantilever up and down using the stage controller to examine the data acquisition (DAQ) output voltage. The stage was positioned under the fixed cantilever using a vernier scale until the cantilever was visually observed to engage with the slide, then the stage was slightly retracted to disengage the cantilever. Next, the Z-direction was adjusted upwards via the pressure-sensitive flexure control system to re-engage the cantilever and bend it within a range of values. A camera mounted above the cantilever recorded the initial cantilever position before bending. As the cantilever tip position shifted, it entered and exited focus. Bending the cantilever downwards was achieved by adjusting the cantilever tip below the microscope coverslip, which was fixed to the stage. When the coverslip was lowered to the top of the cantilever, it forced the tip downwards. The applied stage controller voltage was then plotted against the DAQ response (in voltage converted to nanometers (1V = 10 nm)). The DAQ voltage increased as the cantilever bent upwards. Conversely, the DAQ voltage decreased as the cantilever bent downwards. This is more analogous to the expected response when the curvature of the bridge acceptor IDC forces the cantilever downwards toward the stage.

[0247] Example 11: Optional Flow Pool Components

[0248] refer to Figures 8A to 8BThe flow cell assembly 80 provides an optional method for precisely and repeatably positioning a cantilever-based sensor assembly within a reaction chamber or flow cell, thereby enabling the analyte to interact with the receptor. The flow cell assembly 80 has a substrate 85 having a laminated structure with capillary channels 87 extending between the layers. Channels 87 provide a fluid connection between connectors 82 and 84 extending from the upper surface of the substrate 85 and a test well 86. Connectors 82 and 84 are configured to mate with standard laboratory piping connectors for supplying fluid into the test well 86. Connector 82 provides inflow and outflow of fluid for flushing the test well and sensor system. The fluid used for flushing may be a disinfectant solution, or it may be a solution selected for resetting the sensor, such as an alkaline buffer solution, to dissociate the receptor and analyte. Connector 84 is used to introduce the analyte stream into the test well via a pipette, dropper, or other applicator.

[0249] Figure 9 The diagram illustrates the use of optional flow cell components. Figure 7 A flowchart of the steps of an exemplary pathogen detection process for a system. In step 91, a BCSR is generated using a double-stranded, half-modified DNA bioreceptor, where one strand is modified at both the 5' and 3' ends, while the other strand is unmodified. The BCSR will be used to bridge the gap between the cantilever tip and the base plate of test well 86. (Note that when using integrated sensor 100...) Figure 6E In step 92, the BCSR bridge will span the gap between the cantilever tip and the support base. In step 93, a command is given to the positioning stage 79 via the computer controller to raise the flow cell assembly to create a 150 nm gap between the cantilever tip and the test well bottom plate. Next, in step 94, the dsDNA BCSR is attached to the cantilever tip and the well bottom plate. In step 95, the positioning stage is controlled to lower the flow cell assembly by approximately 5 nm to induce a cantilever deflection. Note that the exact distance is not important. This initial cantilever deflection is used to confirm the successful formation of the initial bridge and for system diagnostics to confirm bridge integrity during testing. Next, in step 96, the dsDNA is denatured by introducing a dissociative agent or other denaturing fluid via conduit 88 and flow channel connector 82. This causes the modified strand to permanently attach to the gap between the cantilever tip and the well bottom plate. Then, in step 97, the unmodified receptor complement is flushed out of the test well. In this sequence, steps 91 through 96 can be grouped into a sequence referred to as “biobridge construction.”

[0250] The following steps constitute the "pathogen detection" sequence. In step 97, a sample containing a 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 includes a suspected receptor complement, the dsDNA will contract or bend due to the IDC, causing the cantilever to be pulled downwards. This deflection of the cantilever will cause the piezoresistive element to change its resistance, which is detected by the detection component and transmitted to the computer controller to output a test result, i.e., a positive test, and store it in computer memory or external memory. If the sample does not contain a suspected receptor complement, no deflection occurs, and the computer controller will report a negative result.

[0251] refer to Figure 8C The exposure of the integrated sensor 100 to the analyte for pathogen detection can be achieved through any of a variety of different methods, including, for example, depositing the sensor 100 at the bottom of the test well 102 and / or attaching it to the bottom of the test well 102. The test well 102 can be a single container or one well in a well array in a multi-well plate, such as those used in high-throughput screening of samples. The test well 102 can be filled with sample processing reagents into which the analyte sample will be introduced. It should also be noted that there are no limitations on the size or dimensions of the reaction chamber or test wells, and any number of integrated sensor assemblies 100 can be placed in the reaction chamber.

[0252] Electrical connector 110 provides connection between the conductor pad on each sensor 100 and the system electronics, as referenced. Figure 7 Although not shown, the bonding pads of the integrated sensor, for example via die attachment, ball wedge bonding, wire bonding, etc., can be connected to a PCB that is connected to a multi-contact socket, multi-pin plug, flexible cable lead, or similar structure to facilitate rapid electrical connection and disconnection of the sensor and system electronics. Analyte 106 can be introduced into test well 102 via dropper 104, pipette, or similar applicator. In some applications, the test well is not configured as a container for holding liquid, but can be an aerosol sample 122 that can be introduced to expose the chamber, vessel, or cuvette 120 of sensor 100, such as... Figure 8D As illustrated in the example. (Note that electrical connections to external devices are not shown.) Aerosol samples can be introduced using methods similar to those used in breath alcohol analyzers. While the fundamental measurement principles differ for detecting alcohol (via the oxidation of ethanol to acetic acid) using a conventional breath alcohol analyzer and for detecting pathogens or other analytes via interaction with receptors of the integrated sensor of this invention, the exposure mechanism is somewhat similar, i.e., the subject can simply exhale air 122 into a mouthpiece 124 connected to the test chamber 120 to allow direct detection of the target's presence by the sensor 100.

[0253] The association between the target analyte within the sample and the biobridge receptor induces a structural change in the bridging molecule (BCSR), which causes the cantilever to deflect. Upon deflection, a piezoresistive element in the cantilever causes a change in the output voltage to generate a detection signal. The test well can have any shape or size, and there is no limitation on the number of biobridge cantilevers that can be used for testing. Alternative structures and methods for exposing the sensor 100 to the analyte to be tested will be apparent to those skilled in the art.

[0254] Example 12: BCSR Sample Processing Reagent (SPR)

[0255] This article describes a sample preparation reagent (SPR) for detecting analytes in a sample. The SPR comprises a buffer, an enzyme activity inhibitor, a denaturant, a low-ionic-strength detergent, and an oxidant. The SPR enables receptors in an integrated sensor to associate with one or more analytes in the sample. The SPR also allows the resulting conformational changes in the receptors to trigger changes in the geometry of the bridging cantilever.

[0256] Buffers in biological systems are designed to maintain intracellular and extracellular pH within a very narrow range and to resist pH changes in the presence of internal and external influences. pH in biological systems controls the solubility, biological function, and chemical reactivity of biological materials. Buffers maintain the pH at which analyte nucleic acids can dissociate from their native complement or intrinsic secondary structures. Non-limiting examples of SPR buffers include phosphates, MES, bis-tripropane, TES, histidine, HEPES, DIPSO, MOBS, TAPSO, Tris, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS. In various embodiments, SPR is between pH 7 and 11.

[0257] Detergents are surfactants with an amphiphilic structure, in which each molecule has a hydrophilic (polar) head and a long hydrophobic (nonpolar) tail. This dual nature of detergents facilitates the mixing of hydrophobic compounds with water, for example, in biological samples.

[0258] SPR detergents can be anionic detergents, cationic detergents, amphoteric detergents, defoaming detergents, or any combination thereof.

[0259] Low-ionic-strength detergents can disrupt cell wall structures to release analytes from cells.

[0260] In some embodiments, the detergent includes Triton X-100, TWEEN-20, NP-40, or Brij-surfactant.

[0261] Oxidizing agents are chemical substances that tend to oxidize other substances, causing them to lose electrons and increase their oxidation state. In SPR, oxidizing agents produce controlled oxidative fragmentation of the analyte.

[0262] In some embodiments, the oxidant includes oxygen, ozone, peroxide compounds, nitrate compounds, nitrite compounds, chlorine compounds, perchlorate compounds, chlorate compounds, chlorite compounds, hypochlorite compounds, bromine compounds, iodine compounds, persulfate compounds, permanganate compounds, chromium compounds, chromate compounds, perborate compounds, bismuthate compounds, copper compounds, iron compounds, cerium compounds, lead compounds, silver compounds, rhenium compounds, hydroxylamine compounds, sulfur oxide compounds, osmium oxide compounds, N-oxide compounds, quinone compounds, amide compounds, imide compounds, cyanurate compounds, saccharin compounds, selenium compounds, perrhenate compounds, propyl gallate, or any combination thereof.

[0263] In specific embodiments, the oxidant includes peroxide compounds, nitrate compounds, permanganate compounds, or any combination thereof.

[0264] In some embodiments, an enzyme activity inhibitor is present in the SPR. Non-limiting examples of useful enzyme activity inhibitors for SPR include chelating agents, RNase inhibitors, DNase inhibitors, protease inhibitors, or any combination thereof.

[0265] Chelating agents are compounds that react with metal ions to form stable, water-soluble metal complexes. The presence of chelating agents in biological samples protects nucleic acids from enzymatic degradation by removing metal ions. Chelating agents also reduce the interaction between proteins and nucleic acids, thus improving the efficiency of nucleic acid extraction from biological samples. Non-limiting examples of SPR chelating agents include EDTA, EGTA, HEDTA, NTA, and TEA.

[0266] RNase inhibitors, DNase inhibitors, and protease inhibitors can also be present in SPR to prevent degradation of analytes during the use of SPR to detect analytes in samples.

[0267] In another embodiment, the SPR further comprises a dissociating agent. The dissociating agent disrupts the hydrogen-bonded network in the aqueous solution, destabilizing the native state of macromolecules (e.g., proteins, nucleic acids) in solution. The dissociating agent denatures nucleic acid-associated proteins, thereby weakening the hydrophobic interactions between the nucleic acid-associated proteins and nucleic acids. The dissociating agent causes the nucleic acids to dissociate from the nucleic acid-associated proteins. Non-limiting examples of dissociating agents include guanidine thiocyanate, guanidine, urea, and thiourea.

[0268] Those skilled in the art will understand and be able to optimize the individual components of the sample preparation reagent (SPR) used to detect analytes in a sample.

[0269] Example 13: Detection of SARS-CoV-2 via biological cantilever deflection

[0270] The 5' and 3' ends of the receptor DNA candidate were chemically modified to enable attachment to the gold surface of the cantilever sensor. Specifically, 5'-phosphothiolization was achieved via a reaction of standard alkaline phosphatase and T4 kinase with ATPγS. The 3' end was then modified to a 3'-thiol-modified oligonucleotide via a T4-ligase reaction. Successful thiol modification was confirmed by electrophoretic analysis 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 formed disulfide-bonded multimers. Prior to bioassembly, the thiol-modified DNA ends were reduced with TCEP to eliminate the thiol-thiol-bonded multimers.

[0271] The cantilever body comprises a piezoresistive material whose conductivity, or resistance, varies with cantilever deflection. The cantilever tip and the supporting surface beneath it are coated with gold to enable bio-attachment to thiol-modified receptors. Twenty silicon piezoresistive MEMS cantilever arms were fabricated with various geometries and physical properties, such as material, length, force constant, and Wheatstone bridge resistance. Initially, two different lengths, 400 nm and 450 nm, and two different tip designs were chosen. The shorter cantilever has a sharp Si tip, while the longer version has no tip. Figure 10 This is a diagram showing the experimental details when using two cantilever arms to test various samples.

[0272] For initial testing, double-sided Kapton tape was used to mount the module onto the silicon wafer. A second sheet of double-sided Kapton tape was used to mask the sensor chip and the support. Only a small section (approximately 1 inch) of the outer plastic protective layer of the tape was later removed. The wafer was placed on the substrate support and loaded into the electron beam evaporation system. Substrate rotation was turned off because the distance between the masking tape and the cantilever tip would cause 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.

[0273] The BCSR bioassembly and detection method involves a series of steps to detect the presence of complementary target molecules using a cantilever with an attach acceptor. This method follows... Figure 9 The general sequence is shown below. First, gold surfaces for the cantilever and support base are prepared, followed by creating a gap between them. Voltage measurements are performed using a data acquisition (DAQ) system. Next, the gap is bridged by introducing a receptor, and an increase in DAQ voltage is observed as the gap size increases, providing evidence of bridging. Unbound complementary strands are removed using a rinsing system, demonstrating the formation of single-stranded DNA bridges, which leads to changes in DAQ voltage. It must be ensured that the voltage differs from the voltage of the unbridged gap before bioassembly. In the detection sequence, a sample containing the complementary target molecule is introduced. The DAQ voltage is measured as an indicator of cantilever deflection due to the binding of the target molecule. Finally, these steps are repeated for additional detection tests.

[0274] To assess the sensitivity and specificity of BCSR component assembly, nine different samples were evaluated using DNA receptor 008. The nine samples are listed below: (1) Positive control – complement: A positive control sample containing approximately 100 copies of 100% complementary DNA in a sample preparation reagent (SPR) solution. (2) Negative control – non-complement: A negative control sample containing approximately 100 copies of 40% complementary DNA in an 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 an SPR solution. (4) COVID-19 virus in SPR: Approximately 1000 copies of heat-inactivated SARS-CoV-2 variant B.1.1.7 (ATCC vr-3326hk) were mixed with an SPR solution. (5) Sputum in SPR: Human sputum 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 sputum-SPR solution. (7) COVID-19 virus in sputum-SPR: Approximately 1000 copies of heat-inactivated SARS-CoV-2 variant B.1.1.7 (ATCC vr-3326hk) mixed with SPR solution. (8) Non-COVID RNA in sputum-SPR: Approximately 1000 copies of genomic RNA from human β-coronavirus OC43 (ATCC VR-1558D) in sputum-SPR solution. (9) Non-COVID virus in sputum-SPR: Approximately 1000 copies of human β-coronavirus OC43 (ATCC VR-1558) in sputum-SPR solution.

[0275] Figure 10This is a bar graph showing the BCSR biocantilever deflection in nanometers (nm) when the nine samples listed above were detected. The BCSR biocantilever deflection (in nanometers (nm)) of the nine samples, both containing and without COVID RNA, was tested 10 times repeatedly. The results showed that positive samples containing COVID RNA produced a BCSR biocantilever deflection of approximately 30 nm, while negative samples lacking COVID RNA showed a deflection of less than 2 nm, a significant difference from the positive samples. The results demonstrate the ability to detect RNA as a potential target analyte in the BCSR sensor. The results also indicate that removing RNA from an RNA-bound BCSR sensor is effective for “resetting” the BCSR sensor. Legend in the figure: Complement = 100% complementary target, Non-complement = Target material with only 80% homology, COVID RNA = Purified RNA from the target virus, COVID virus = Target COVID SARS CoV-2 virus, Sputum = Human sputum analogue without target material, Non-COVID = Non-COVID-19 coronavirus. The baseline or physical dissociation of the BCSR cantilever deflection is always approximately 1 nm.

[0276] To further evaluate the reliability and reproducibility of BCSR component assembly, three different cantilevers (black bar, gray bar, and diagonal line) with DNA receptor 008 were tested to detect four types of analytes (precise nucleic acid receptor complement, non-complementary nucleic acid, genomic COVID 19 RNA complement, and non-COVID coronavirus genomic RNA). Figure 11 The results shown indicate that all three BCSR sensors produce approximately the same cantilever deflection. Although the spring constants of each cantilever differ slightly, the positive and negative detection results are consistent. These results demonstrate that the BCSR biocantilever is reliable and can be manufactured consistently and robustly.

[0277] Figure 12This table illustrates the correlation between the intrinsic curvature of the receptor and the magnitude of the BCSR sensor response. Three DNA receptors (008, 012c, and 012s) were designed, and hybridization tests were performed against complementary DNA. The difference between 012c and 012s is a single nucleotide variation. In the hybridization of DNA receptor 008 with its complementary DNA, the maximum calculated reduction in end-to-end distance was 48 nm. Hybridization of receptor 012c with its complement reduced the distance to 62 nm, while hybridization of receptor 012s with its complement reduced the distance to only 27 nm. Three-dimensional projections (XZ and XY perspective views) show the relative predicted curvature for each receptor-complement pair. In contrast, the measured cantilever deflections for receptors 008, 012c, and 012s were 28 nm, 32 nm, and 17 nm, respectively. In each case, the magnitude of the cantilever deflection was directly correlated with the calculated amount of intrinsic molecular curvature at 58% ± 5%. It is important to note that complete hybridization between complementary strands will be inhibited by the fact that the bridging acceptor strand is permanently attached to the cantilever and the platform at both ends, respectively.

[0278] Example 14: Detection of SARS-CoV-2 via biological cantilever deflection

[0279] Testing was conducted to evaluate the ability to detect multiple biological targets, including COVID, influenza (H1N1), adenovirus, and human rhinovirus (HRV). Specifically, the following receptors were targeted: COVID, H1N1 2c, H1N1 3a, adenovirus_41, adenovirus_49, adenovirus_52, HRV A_12, HRV B, and HRV C.

[0280] Figure 13 The tables in the table provide detailed information on the design and production of receptors for detecting influenza A virus, adenovirus B, and human rhinovirus (HRV). Two (2) to three (3) different candidate receptors were developed for each target. This redundancy allows for the selection of the receptor with the greatest detection performance, or for the selection of a multiplex system that detects responses based on multiple biomarkers of the target genome. As described in the table, many biophysical properties were considered.

[0281] Figure 13The columns of the table shown are defined as follows: Receptor "Source": Common regions shared by the most predominant variant of a viral fragment, a specific NCBI / NIH database accession number, or a target. (Data for each identified source can be readily found by those skilled in the art at the World Wide Web ncbi.nlm.nih.gov, which is incorporated herein by reference.) "Candidate": Internal designation of a specific receptor candidate. "C&S": Relative measure of receptor curvature and stiffness – larger values ​​are directly associated with a higher detection response. "ENDS": A measure of receptor curvature, such as the ratio of the actual length of the receptor along its helical axis to the end-to-end distance – larger values ​​are directly associated with increased curvature. "Reduction %": A more intuitive measure of receptor curvature, such as the actual molecular length compared to the end-to-end distance – larger values ​​are directly associated with increased curvature. The reduction % can range widely, for example from approximately 15% to close to 80%. “ΔG”: Gibbs free energy is a measure of the internal folding potential of the receptor molecule – larger values ​​are associated with fewer problems with the internal folding of the target genomic marker, thus inhibiting detection. Tm℃: A calculated measure of the unwinding temperature of the target genomic fragment, which is critical for proper denaturation and hybridization with the receptor. “Target Homology”: The calculated genetic match between the receptor and its target genomic fragment – ​​higher homology infers greater specificity. “Maximum Homology with Non-Target %”: The percentage of match with the closest genetic variant, neighboring, or other microorganisms that may be found in the sample – lower values ​​infer greater specificity.

[0282] The identified targets were selected to determine the effectiveness of the method of the present invention in differentiating between various biological agents that induce cold and flu symptoms. Receptors were designed based on necessary biophysical properties, including high specificity for the desired target and low homology with genetic neighbors. Figures 14A to 14I The bar graph results provided show the responses from five (5) piezoresistive cantilever devices (solid or striped bars), each tested 10 times and reset between each test. The reset responses consistently showed approximately 5 nm + / - 1 nm, as expected in the experimental design for all tests. The vertical scale (nanometers) was all adjusted to 100 nm for graph-to-graph comparisons. Note that the deviation of the response to a single target biodetection (i.e., cantilever deflection) is proportional to the calculated and independently validated intrinsic curvature of the receptor. For example, curvature-induced reduction in the end-to-end distance of the H1N1 2c receptor ( Figure 14B The predicted value is approximately 81 nm. The measured cantilever deflection is approximately 50 nm. On the other hand, the value predicted by the HRV-C receptor ( Figure 14IThe predicted cantilever deflection was 53 nm, while the measured deflection was 33 nm. In each case, the measured cantilever deflection was approximately 60% to 65% of the predicted reduction in receptor length. This observation is not surprising, given that the structural change was less than expected due to the incomplete formation of the double helix.

[0283] While the degree of deflection may not reach the predicted level, the deflection is reproducibly proportional to the predicted curvature and has sufficient amplitude for reliable detection. At least ten tests were performed on each sample using each of the receptors across multiple cantilever arms. The results between tests were also highly consistent. In summary, the binary results of the IDC detection technology have been demonstrated to accurately and consistently detect the virus 10 out of 10 times within seconds in the presence of sputum simulants.

[0284] Example 15: Other Implementation Methods

[0285] Project 1. A sensor assembly for detecting a target analyte, the sensor assembly comprising:

[0286] A cantilever made of silicon material, having a deflection detection element disposed therein, the cantilever being elongated and having a proximal end and a distal end;

[0287] A support base having a silicon substrate disposed on a support surface;

[0288] A first conductive metallization layer is disposed on the lower surface of the cantilever and is in electrical contact with the deflection detection element;

[0289] A second conductive metallization layer is disposed on the upper surface of the silicon substrate, and the second conductive metallization layer is configured to conduct electrical signals between the deflection detection element and a contact electrically connected to an external electrical device.

[0290] A eutectic bond is formed between a first conductive metallization layer and a second conductive metallization layer and the substrate, the eutectic bond being configured to secure the proximal end of the cantilever to the substrate, wherein the substrate has a substrate thickness to support the lower surface of the distal end of the cantilever at a fixed gap from the support surface; and

[0291] At least one bridging acceptor having a first end and a second end, wherein the first end is configured to attach to the lower surface of the distal end of the cantilever, and the second end is configured to attach to the support surface, wherein the bridging acceptor is configured to change conformation and cause deflection of the cantilever when interacting with the target analyte.

[0292] The deflection detection element generates an output signal indicating the deflection of the cantilever.

[0293] 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.

[0294] 3. The sensor assembly according to Item 2, wherein the plurality of varistors are configured to define a Wheatstone bridge.

[0295] 4. The sensor assembly according to Item 1, wherein the eutectic bonding comprises gold-silicon bonding.

[0296] 5. The sensor assembly according to Item 1, wherein the fixed gap is in the range of 1 to 1,000 nm.

[0297] 6. The sensor assembly according to Item 1, wherein a metallized contact region is formed on each of the lower surface and the support surface at the distal end of the cantilever for attaching the end of the at least one bridge acceptor.

[0298] 7. The sensor assembly according to item 6, wherein the metallized contact region comprises gold, and the at least one bridge acceptor is modified with thiol to facilitate attachment to the metallized contact region.

[0299] 8. The sensor assembly according to Item 1, wherein the at least one bridging acceptor 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 of the molecules, a conjugate of any of the molecules, a hybrid of any of the molecules, or any combination thereof.

[0300] 9. The sensor assembly according to Item 8, wherein the at least one bridging acceptor comprises nucleic acid, nucleic acid analog, double-stranded DNA (dsDNA), dsDNA analog, single-stranded DNA (ssDNA), ssDNA analog or peptide nucleic acid.

[0301] 10. The sensor assembly according to item 8, wherein the at least one bridging acceptor is ssDNA.

[0302] 11. The sensor assembly according to Item 7, wherein the at least one bridging acceptor is a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

[0303] 12. The sensor assembly according to item 7, wherein the length of the at least one bridge acceptor is 50 to 1,000 nm.

[0304] 13. The sensor assembly according to Item 7, wherein the bridge acceptor experiences a length reduction of from about 15% to about 80% when interacting with the target analyte.

[0305] 14. The sensor assembly according to any one of items 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 of the molecules, conjugates of any of the molecules, hybrids of any of the molecules, or any combination thereof.

[0306] 15. The sensor assembly according to Item 14, wherein the analyte comprises complementary nucleic acid, complementary nucleic acid analog, complementary dsDNA, complementary dsDNA analog, complementary ssDNA, complementary ssDNA analog, complementary RNA or complementary RNA analog.

[0307] 16. The sensor assembly according to item 14, wherein the analyte is ssDNA.

[0308] 17. The sensor assembly according to item 14, wherein the analyte is RNA.

[0309] Project 18. An analyte detection system, comprising:

[0310] A test well or test chamber configured to receive a sensor assembly according to any one of items 1 to 13; and

[0311] A conductive connector is configured to enable electrical communication between the sensor assembly and the instrument to generate an external display indicating the detected deflection of the cantilever.

[0312] 19. The analyte detection system according to Item 18, wherein the test well or test chamber is configured to retain samples, said samples including one or more of the following: tears, saliva, oral fluid, bronchoalveolar lavage fluid, mucus, nasal samples, nasopharyngeal samples, respiratory samples, urine, feces, tissue, blood, plasma, serum, cell culture, body fluid, tissue biopsy, apocrine fluid, or exocrine fluid, forensic samples, aerosols, soil, water samples, food, ingredients, raw materials, process samples, by-products, products, or quality control samples.

[0313] 20. The analyte detection system according to Item 19, wherein the sample includes viruses, bacteria, bacteriophages, yeast, mycoplasma, fungi, human cells, animal cells, plant cells, insect cells, or any combination thereof.

[0314] 21. The analyte detection system according to Item 19, wherein the sample is a mucus sample, nasal sample, respiratory sample, or nasopharyngeal sample.

[0315] 22. The analyte detection system according to Item 19, wherein the sample contains a virus.

[0316] 23. The analyte detection system according to Item 18, wherein the virus is one or more of coronavirus, influenza virus, respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or Zika virus.

[0317] The integrated NEMS cantilever sensing device and method disclosed herein provide an accurate, reproducible, and cost-effective tool for detecting pathogens and other biomarkers. The sensor unit can be fabricated at extremely low cost, in dimensions of 100 micrometers, and in fractions of a milligram using established MEMS fabrication techniques and processes. Using the described exemplary fabrication process, large-scale, reliable, consistent, and reproducible detection units with high specificity can be manufactured and used to detect analytes without the need for separation and purification processes. Optional reconfigurable flow cell embodiments offer significant versatility for applications in clinical settings and for large-scale sample analysis, as may be encountered in agricultural, food and water safety, and public health settings such as schools and ports of entry. Unlike many existing diagnostic tests in which detection reagents have a limited lifespan, many elements of the method of the present invention can be stored (e.g., MEMS and electronic components) for extended periods without the risk of degradation. The ability to rapidly sequence and synthesize DNA makes the method of the present invention a realistic and cost-effective tool for preparing for detection and containment in the event of future infectious diseases.

Claims

1. A sensor assembly for detecting a target analyte, the sensor assembly comprising: a cantilever formed of a silicon material having a deflection detection element disposed therein, the cantilever being elongate 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 layer disposed on a lower surface of the cantilever in electrical contact with the deflection detection element; a second conductive metallization layer disposed on an upper surface of the silicon pedestal, the second conductive metallization layer configured to conduct an electrical signal between the deflection detection element and a contact in electrical communication with an external electrical device; a eutectic bond formed between the first and second conductive metallization layers and the pedestal, the eutectic bond configured to secure the proximal end of the cantilever to the pedestal, wherein the pedestal has a pedestal thickness to support a lower surface of the distal end of the cantilever at a fixed gap from the support surface; and at least one bridge acceptor 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, wherein the bridge acceptor is configured to change conformation and cause deflection of the cantilever upon interaction with the target analyte; wherein the deflection detection element generates an output signal indicative of the deflection of the cantilever.

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

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

4. The sensor assembly of claim 1, wherein the eutectic bond comprises a gold-silicon bond.

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

6. The sensor assembly of claim 1, wherein a metallized contact area is formed on each of the lower surface of the distal end of the cantilever and the support surface for attachment of an end of the at least one bridge acceptor.

7. The sensor assembly of claim 6, wherein the metallized contact area comprises gold and the at least one bridge acceptor is thiol-modified to facilitate attachment to the metallized contact area.

8. The sensor assembly of claim 1, wherein the at least one bridge acceptor 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 of the molecules thereof, a conjugate of any of the molecules thereof, a hybridization of any of the molecules thereof, or any combination thereof.

9. The sensor assembly of claim 8, wherein the at least one bridge acceptor comprises a nucleic acid, a nucleic acid analog, double-stranded DNA (dsDNA), a dsDNA analog, single-stranded DNA (ssDNA), a ssDNA analog, or a peptide nucleic acid.

10. The sensor assembly of claim 8, wherein the at least one bridge acceptor is ssDNA.

11. The sensor assembly of claim 7, wherein the at least one bridge acceptor is a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units connected by peptide bonds.

12. The sensor assembly of claim 7, wherein the at least one bridge acceptor is 50 to 1,000 nm in length.

13. The sensor assembly of claim 7, wherein the bridge acceptor undergoes a length reduction of from about 15% to about 80% upon interaction with the target analyte.

14. The sensor assembly of any one of claims 1 to 13, wherein the analyte comprises 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 of its molecules, a conjugate of any of its molecules, a hybridization of any of its molecules, or any combination thereof.

15. The sensor assembly of 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 of claim 14, wherein the analyte is ssDNA.

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

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

19. The analyte detection system of claim 18, wherein the test well or test chamber is configured to retain a sample comprising one or more of a tear, a saliva, a buccal fluid, a bronchoalveolar lavage fluid, a mucus, a nasal sample, a nasopharyngeal sample, a breath sample, a urine, a stool, a tissue, a blood, a blood plasma, a blood serum, a cell culture, a bodily fluid, a tissue biopsy, a cerumen, or an eccrine or apocrine gland secretion, a forensic sample, an aerosol, a soil, a water sample, a food, an ingredient, a raw material, an in-process sample, a byproduct, a product, or a quality control sample.

20. The analyte detection system of claim 19, wherein the sample comprises a virus, a bacterium, a bacteriophage, a yeast, a mycoplasma, a fungus, a human cell, an animal cell, a plant cell, an insect cell, or any combination thereof.

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

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

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

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