Integrated MEMS sensor with bridged conformational shifting receptors

Abstract

An integrated NEMS cantilever sensor with a precisely controlled cantilever gap has a Receptor attached to bridge the gap. When the Receptor interacts with a target analyte, it changes conformation, inducing a change in the gap. Detectors within the sensor generate signals corresponding to the change in gap dimensions, confirming interaction with the analyte.

Description

[0001] INTEGRATED MEMS SENSOR WITH BRIDGED CONFORMATIONAL SHIFTING RECEPTORS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 488,865, filed March 7, 2023, which is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The present application generally relates to devices with Micro-Electro-Mechanical Systems (MEMS) sensors and more specifically to devices and methods for detection, identification, and quantification of target analytes.

[0006] BACKGROUND

[0007] State of the art analyte diagnostic technologies employ fundamental chemistry and analytic instrumentation such as spectrophotometry, gas or liquid chromatography. Within the context of this discussion, an analyte is defined as any inorganic or organic compound or material about which information is sought, which may include identity, purity, quantity and / or concentration. For example, currently employed pathogen sensing technologies utilize enzyme-based immunoassays, polymerase chain reaction (PCR), fluorescence signaling, or quartz microbalance and silicon microcantilevers to determine the presence and / or mass of the target pathogen. Unfortunately, state-of-the-art technologies often lack the speed, accuracy, and sensitivity to provide adequate informed decision support for the end user. The presence of background materials typically detracts from the accuracy of these technologies and thus requires extensive sample preparation prior to diagnosis. Further, these technologies are so cumbersome and complicated to operate that they are prone to false positive and negative results.

[0008] Addressing the needs of the modern analyte diagnostic market means developing technology that is sensitive enough to accurately detect relatively few molecules, as rapidly as possible, and with an ambivalence to environmental background materials. New materials, nanotechnology, and miniature bio-mechanical devices have enabled significant progress toward this objective.

[0009] Micro-Electro-Mechanical 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 technology. MEMS have several distinct advantages as a manufacturing technology including that the interdisciplinary nature of MEMS technology and its micromachining techniques, as well as its diversity of applications, has resulted in an unprecedented range of devices and synergies across previously unrelated fields, e.g., biology and microelectronics. In addition, MEMS batch fabrication techniques allow components and devices to be manufactured with increased performance and reliability in a configuration having reduced physical size, volume, weight, and cost. Further, MEMS provide a basis for the manufacture of products that cannot be made by other methods. These factors make MEMS potentially a far superior detection technology.

[0010] Currently, most MEMS applications are found in systems ranging across automotive, electronic, communication, and defense applications. Their unique mechanical properties and miniaturized sizes make MEMS sensors ideal for use, inter alia, as accelerometers for airbag sensors, inkjet printer heads, computer disk drive read / write heads, projection display chips, blood pressure sensors, optical switches, and microvalves.

[0011] Improvements in MEMS processes technologies have greatly facilitated biosensor developments in medical applications. Such improvements include bulk surface micromachining that selectively removes parts of the silicon or adds additional structural layers to form the mechanical and electromechanical components. MEMS based biosensors can not only exploit the electrical properties of silicon, but also take advantage of either silicon’s mechanical properties and / or both its electrical and mechanical properties.

[0012] Cantilevers are one of several fundamental components of MEMS with dimensions in the micro- or nano-range. Cantilevers may be fabricated from silicon (Si), silicon nitride (SisN4), 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 free-standing beam, anchored at one end. Cantilevers can be fabricated as a single unit or as an array of multiple units and can be designed with different degrees of "stiffness" or flexibility to meet the requirements of specific applications. These processes also allow for the fabrication and integration of the electronic circuitry and other MEMS components required to interface with the cantilevers. Their flexibility and versatility make them a promising component for use in a variety of environmental, biomedical, and consumer product applications. Prior art biosensing applications often integrate MEMS cantilevers within test chambers to isolate the receptor - analyte reaction to a minimal volume. In conventional MEMS sensor cantilevers, bending in both static and dynamic modes is accomplished through alterations in mass and / or the Young's modulus. The dynamic mode utilizes oscillation as a driving force, as opposed to gravity in the static mode. However, these conventional MEMS cantilevers have significant limitations.

[0013] Cantilever sensors can be operated in static mode and / or in dynamic mode. In static mode, molecular interactions on the cantilever surface are translated into a cantilever bending as a result of changes in the surface stress. In static mode, the displacement or deflection in the cantilever can be measured using different techniques such as diffracted or reflected light, or piezoresistive materials. In dynamic mode, a change in mass, or Young’s modulus, can be measured by the resonant frequency of the oscillating cantilever. The Young’s modulus can induce a negative or positive frequency shift depending on the thickness of the deposited layer of analyte. Dynamic mode is more sensitive to changes in the cantilever characteristics than is the static mode. It should be noted that for biocantilever devices known in the art, a sufficient mass must be deposited on the device to affect a detectable response. Additionally for such bio-cantilever devices, non-specific deposition from background materials is a known challenge and requires cantilever surface masking or sample preparation or purification to achieve sufficient specificity.

[0014] Dynamic Mode can be described in the following mathematical equations.

[0015] Equation of force balance for a cantilever is: where Aus the spring constant and yo is the neutral axis. To obtain optimal performance due to a change in mass, it is assumed that there is no damping (y=0), a simple linear spring, and transient force has been applied, Fext= 0. If Ay = (y0— y) Equation (1) simplifies to: mf!!fcW+ / ; (Ay)= 0 (2)

[0016] Solving for the derivative yields, Ay = Aeia)ot, where m0is the fundamental natural oscillation frequency of the cantilever.

[0017] The resonance frequency of the cantilever before any mass is added is and after mass is added is where ma= mb+ Am and ka= mkb+ Afc.

[0018] Modifying Equation (3) by taking the logarithm and then differentiating provides

[0019] Equation 5: which shows the resonance frequency shift is a function of both the change in mass Am and a function of change the spring constant Afc.

[0020] If the spring constant is unchanged during the mass deposition, then the change in frequency is a function of the change in deposited mass so that Equation 5 simplifies to

[0021] Am = mb(6)

[0022] Substituting from above, Equation 6 becomes

[0023] The associated mass of the cantilever before adding mass is the volume

[0024] Vbmultiplied by density p and a geometric coefficient a . For a straight beam clamped at one end, a = 0.24.

[0025] Vb= LWH and mb= a^ pWLH.

[0026] Using these relationships, Equation (7) becomes

[0027] The change in resonance frequency is directly proportional to the change in mass multiplied by a collection of constants that define the characteristics of the cantilever.

[0028] If the change in mass loading on the cantilever is negligible compared to the in the spring constant, Equation (5) simplifies to

[0029] . , EWH3where k = — —

[0030] 4-L3

[0031] Determination of the change in the spring constant of the cantilever as a function of the height of analyte deposited or adsorbed onto the surface is accomplished by taking the differential of k with respect to H in equation 9, which provides an approximation for small A / E The additional analyte essentially modifies the Young’s modulus or cantilever stiffness. where dH NsAtHt, Nsis the number of analyte components on the surface of the cantilever, Atis the surface covered by the analyte component, and Htis the height of the individual analyte components. For typical applications reported in the literature, the cantilever constant can be changed on the order of 5% and up to 40%.

[0032] The cantilever’s response to a change in mass relative to the change in Young’s modulus can be calculated. The point at which the relative change in frequency due to mass loading (negative) and Young’s modulus (positive) contribute are equal. The condition is satisfied when there is no apparent resonance frequency shift due to addition of analyte component on the cantilever surface, i.e.,

[0033] The mass of the cantilever before and after mass addition are, respectively, mb= a pbWLH, and ma= a- PhWL H + d lc), where Hcis the critical height of the analyte component layer.

[0034] The spring constant k before additional analyte material is added from Equation (9) above. After addition of analyte, the spring constant becomes j _ a2EW(H+dH~)3, .

[0035] 12L3such that where Ebis the Young’s modulus of the cantilever before analyte component is added, Eais the Young’s modulus of the cantilever after addition of analyte component is added, pbis the density of the cantilever before addition of analyte component, and pais the cantilever density after addition of analyte component. Typical values for a cantilever can yield a dHcon the order of 20nm to 30nm.

[0036] MEMS cantilevers in aqueous solutions, i.e., not in air, raise additional challenges for detection due to the significant damping effect on the dynamic motion of the cantilever, where y » 0. The solution for Equation 1 becomes, y = Ae~au,otsin (m t + 0) (14) where Q is the quality factor, and m' = S - -2 ), (16) y m 4mzwhere a> is the dampened resonance frequency of the cantilever in aqueous solution. Aqueous solution also has an effect of frequency peak broadening compared to a cantilever in air with a very low damping coefficient, y ~ 0. This puts the upper limit of detection in the picogram range.

[0037] Static mode detection does not induce an oscillation such that no acceleration or velocity terms are introduced into the system for the force balance equations. For typical cantilever springs this is on the order of multiple 10,000 Gs at resonance frequency in the MHz range. In the static mode the force on the cantilever is from the gravitation field of the earth, where the force is simply F = mg, where g is gravitational acceleration.

[0038] Thus, the force balance equation for a cantilever operating in the static mode is simplified to ky = Fext= mg.

[0039] Taking the logarithm and differentiating yields

[0040] Afc ! Ay _ Am k0y0m0’ or for displacement

[0041] As in the dynamic mode, the static mode system displacement is a function of both change in mass and change in the spring constant.

[0042] For a cantilever spring system where the spring constant does not change, Equation (18) can be rearranged in terms of the amount of displacement due to the addition of mass, i.e.,

[0043] Ay = Am- (19) k

[0044] Because the change in total mass due to typical mass loading on a MEMS cantilever is extremely small, cantilever displacement or deflection is extremely small — on the order of femtometers (fin). This magnitude of displacement is not amenable to known means of transduction. Without the acceleration and velocity term contributing to the equation of motion, the pure static mode of detection is significantly inferior to the dynamic mode of detection.

[0045] The relative change in spring constant to effect displacement when relative mass loading is small compared the spring constant change is Modification of the Young’s modulus, due to surface material modification, independent of mass loading, is very difficult to measure. The small displacements associated with a method of a pure static mode is therefore not amenable to a detection scheme.

[0046] Nonetheless, the static mode can be improved by employing additional forces to effectively reduce the apparent cantilever spring constant. Adding an electric force via capacitor configuration can significantly improve the response to changes in mass loading, but modifying the effective spring constant has its limits due to the very non-linear behavior of the dynamic electric of the change plate distance (cantilever to base gap).

[0047] The cantilever surface can be further coated or functionalized to enhance performance. In order to probe a specific analyte in a sample, the surface of the cantilever may be coated or functionalized with a layer of specific chemical or biological molecules. The surface functionalization of cantilevers is normally undertaken after the sensor is fabricated because the harsh depositing and etching processes can damage any pre-existing functionalization. The functional layer 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 will directly influence the performance of the sensor signal.

[0048] Cantilever surface modifications for chemical, biological, and biomedical applications fall into two categories: physical and chemical. Physical modification results in a change in the topography or morphology of the surface with little to no change in the chemistry, such as etching, grit-blasting, and machining. Well-established chemical techniques include plasma and chemical vapor deposition, atomic layer deposition, and electro-chemical deposition. Chemical treatment can result in oxidation / nitriding / carbiding a surface, surface functionalization, ion infusion, single layer coatings, or coatings comprising many layers of different compositions. Goals of modifying the surface of a cantilever can include to (1) produce specific surface topographies; (2) improve biocompatibility; (3) modify surface composition; (4) create a layer of substances with specific chemical composition.

[0049] Many tools have been developed to solve the challenges of cantilever surface functionalization. Microcapillaries are relatively simple to use and are suitable for functionalizing cantilevers in small quantities. Micromanipulators with translational stages precisely position a microcapillary tubing filled with chemicals to approach a cantilever sensor. Capillary tubing enables certain cantilever sensors to contact a chemical solution for functionalization. Another efficient method of functionalizing cantilevers is to use a chemical inkjet printing technique, which can be used to fabricate high density DNA and protein microarrays. The functionalization of self-assembled monolayers, polymer solutions, and DNA samples has been demonstrated with comparable performance on cantilever sensors. Using this method, microcantilever sensors can be batch functionalized at the wafer level. Other potential variations of the cantilevers are known, for example, the cantilever surface can be modified to form one or more porous silicon region that exhibits different morphological, chemical, thermomechanical, and photonic properties. One example of porous silicon processing is provided in U.S. Patent No. 7,433,811 of Gao, et al. which is incorporated herein by reference.

[0050] Further expanding on the use of MEMS as sensors are biological microelectromechanical systems: Bio-MEMS, which involve the use of small biological- mechanical structures that respond to changes in the environment. These changes may include the introduction of an organic or inorganic analyte, or thermal changes in the environment. The biological component of Bio-MEMS, called the “Receptor”, responds to the environmental change, and this response can be translated into movement of the mechanical structure.

[0051] The related art in Bio-MEMS sensors includes a variety of device designs, modes of signal generation and transduction, and orientation of biological components. In general, the top surface of a MEMS cantilever device is coupled to Receptor molecules that specifically bind to a unique Target molecule. For example, immobilized antibody Receptors would interact with specific antigen Targets. The Receptor-Target complex thus significantly changes the mass of the MEMS device, resulting in a change in orientation, deflection, or change in resonance of the MEMS. Bio-MEMS may operate in either static or dynamic modes. In static mode, the association of Target molecules to the Receptor- coated surface induces stress that induces bending of the cantilever. In dynamic mode, the resonance frequency of the cantilever decreases as Target molecules adsorb onto the Receptor-coated surface. Either approach to detection requires a substantial number of Receptor-Target complex molecules to induce significant changes in the position and / or resonance of the MEMS sensor. These techniques can lack specificity, demonstrate very poor sensitivity, and are extremely difficult to consistently manufacture at large scale. A means of signal transduction or measuring cantilever deflection may be interferometry or optical beam deflection off the cantilever free end, which is typically seen in AFM (atomic force microscopy) instruments. This approach requires a laser light source and position sensitive photodetector which tend to require frequent calibration and consistent sample refractive indices. As a result, this approach often fails in applications outside of a controlled laboratory environment. Another common approach may be to apply piezoresistive materials to the cantilever, whereby stress induces a change in conductivity and / or resistance.

[0052] In other prior art, various molecules are envisioned to bridge the gap between opposing electrodes. For example, US8,078,408 and US9,857,366 of Albert, et al. (incorporated herein by reference), US10,036,064 and US2019 / 0094175 of Merriman, et al., teach bridged electrodes that complete a circuit and / or inhibit resonance as a result of electrically conductive, high A / G composition DNA. In the Merriman disclosures, association of the bridged molecule with molecules in its environment does not induce mechanical deflection of the electrode(s). The ‘408 and ‘366 patents of Albert describe bridging a gap between free ends of two surfaces with a template molecule of ssDNA to form a circuit. Upon hybridization with a target molecule, the dsDNA increases conductivity across the circuit. While the written description mentions an alternative approach for measuring cantilever deflection using piezoresistance, the absence of an enabling disclosure combined with the inventors’ subsequent inability to fabricate a functional device with any degree of repeatability in the gap and behavior led to abandonment of the effort.

[0053] Among the abundant variety of molecular interactions is nucleic acid hybridization, which occurs when single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal and hybridize to complementary DNA or RNA molecules. Related methods and devices for detection of biological Targets have applied the fundamental principle of hybridization between complementary nucleic acid base pairs, e.g., cytosine bonding to guanine, and adenine bonding to thymine or uracil. Methods are known whereby a specific sequence of nucleotides, typically bound to a radioactive, fluorescent, or chromogenic tag, is exposed to an array of nucleic acid molecule fragments covalently attached to a surface or material such as nylon or nitrocellulose. If the nucleotide sequence of the tagged “probe” is complementary to a fragment within the array of molecules bound to the surface, hybridization occurs, and detection is determined by the presence of the radioactive, fluorescent, or chromogenic signal. In general, the aforementioned transduction technologies of the hybridization require significant quantities of analyte, resulting in a reduced lower limit of detection.

[0054] Sensing methods and devices are known that exploit intrinsic or inherent molecular structure and changes in molecular structure as a result of changes in the environment surrounding the molecule. Examples of environmental influence on molecular structures are changes in temperature, pH, salinity and light emission or absorption. Other examples of environmental influence include the introduction of other molecules that, through various interactions (i.e., molecular exclusion, hydrophilicity, ionic bonding, etc.), change the structure of the resident molecules. As an example, neural proteins called prions can convert from alpha helix to beta sheet conformation in the presence of other beta sheet prions. As another example, the introduction of single-stranded DNA molecules into the presence of complementary DNA strands can result in hybridization to form a double-stranded DNA helix. Known in the art is the potential of double-stranded DNA molecules to exhibit inherent curvature that serves critical biological functions such as nucleosome positioning and genetic expression. Various theoretical models have been proposed to explain the relationship between DNA nucleotide sequence and deflection of the helical axis or inherent DNA curvature. Common among these models is the number, position, and length of tracts of adenine nucleotides called A-tracts. The hydrogen bonds between guanine and cytosine residues are perpendicular to the helical axis. Conversely, the bonding between adenine and thymine residues has a pronounced 2° - 4° angle per base pair. Thus, A-tracts positioned on the same side of the DNA helix are able to induce an inherent curvature of the molecular axis, significantly reducing the end-to-end distance of double stranded nucleotide sequences.

[0055] SUMMARY

[0056] Devices and methods disclosed herein are directed to detection of virtually any analyte, regardless of the sample type or background materials within the sample. In some embodiments, the devices comprise one or a plurality of integrated sensors, which sensors are configured to form a bridged cantilever geometry. The integrated sensors comprise a first surface, a second surface, one of which is flexible, and a Receptor. The Receptor comprises one or a plurality of molecular structures that change conformation upon interaction, association, or disassociation with one or a plurality of analytes. A first point of contact on the receptor is attached to a first surface contact point on the first surface and a second point of contact on the receptor is attached to a second surface contact point on the second surface. In some embodiments, the receptor is in a physically analyte-associated configuration when the receptor is associated with the analyte. In some embodiments, the receptor is in a physically analyte-disassociated configuration when the receptor is not associated with the analyte. The detector generates an output signal when the receptor interacts, associates, or disassociates with the analyte.

[0057] Also provided is a sample processing reagent (SPR) and a method of using SPR for detecting an analyte in a sample. The SPR comprises a pH buffering component, an enzymatic activity inhibitor, a denaturant, a low ionic strength detergent, and an oxidizing agent. The buffering component maintains a pH that supports disassociation of analyte from its ligand, native complement, or inherent secondary structure. The low ionic strength detergent disrupts host viral or cell wall structures to release the analyte from a cell. The oxidizing agent produces a controlled oxidation fragmentation of the analyte.

[0058] In one aspect, a sensor assembly for detecting a target analyte includes: a cantilever formed from a silicon material having a deflection detection element disposed therein, the cantilever being elongated and having a proximal end and a distal end; a support base having a silicon pedestal disposed on a support surface; a first conductive metallization disposed on a lower surface of the cantilever in electrical contact with the deflection detection element; a second conductive metallization disposed on an upper surface of the silicon pedestal, the second conductive metallization configured to conduct electrical signals between the deflection detection element and contacts in electrical communication with external electrical devices; a eutectic bond formed between the first and second conductive metallization and the pedestal, the eutectic bond configured to secure a proximal end of the cantilever on the pedestal, wherein the pedestal has a pedestal thickness to support a lower surface of the distal end of cantilever at a fixed gap from the support surface; and at least one bridge receptor having a first end and a second end, wherein the first end is configured for attachment to the lower surface of the distal end of the cantilever and the second end is configured for attachment to the support surface, wherein the bridge receptor is configured to change conformation upon interaction with the target analyte and induce deflection of the cantilever; wherein the deflection detection element generates an output signal indicative of deflection of the cantilever.

[0059] In some embodiments, 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. The plurality of piezoresistors may be configured to define a Wheatstone bridge. The eutectic bond may be a gold- silicon bond. In some embodiments, the fixed gap is within a range of 1 - l,000nm. A metallized contact area may be formed on each of the lower surface of the distal end of the cantilever and the support surface for attachment of the ends of the at least one bridge receptor. The metallized contact area may be gold, where the at least one bridge receptor is thiol-modified to facilitate attachment to the metallized contact areas.

[0060] The at least one bridge receptor may be an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof. In some embodiments, the at least one bridge receptor may be a nucleic acid, a nucleic acid analogue, a double-stranded DNA (dsDNA), a dsDNA analog, a single-stranded DNA (ssDNA), a ssDNA analog, or a peptide nucleic acid. The at least one bridge receptor may be a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The at least one bridge receptor may be 50 - 1,000 nm in length and may further undergo reduction in length from about 15% to about 80% upon interaction with the target analyte.

[0061] In some embodiments, the analyte detected by the sensor assembly may 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 repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof. The analyte may be a complementary nucleic acid, a complementary nucleic acid analogue, a complementary dsDNA, a complementary dsDNA analog, a complementary ssDNA, a complementary ssDNA analog, a complementary RNA, or a complementary RNA analog. The analyte may be ssDNA or it may be RNA.

[0062] In another aspect, an analyte detection system including a test well or chamber configured to receive the sensor assembly as described above and conductive connectors configured for electrical communication between the sensor assembly and instrumentation for generating an external display indicative of detected deflection of the cantilever. The test well or chamber may be configured to retain a sample of one or more of a lachrymal fluid, saliva, a buccal fluid, a bronchoalveolar lavage fluid, mucus, a nasal sample, a nasopharyngeal sample, a breath sample, urine, feces, tissue, blood, plasma, serum, a cell culture, a bodily fluid, a tissue biopsy, an apocrine fluid, or an eccrine fluid, a forensic sample, an aerosol, soil, water sample, food, an ingredient, a raw material, an in-process sample, a byproduct, a product, or a quality control sample. The sample may include a virus, a bacterium, a phage, a yeast, a mycoplasma, a fungus, a human cell, an animal cell, a plant cell, an insect cell or any combination thereof. The sample may be a mucus sample, a nasal sample, a breath sample, or a nasopharyngeal sample. The sample may contain a virus, which may be one or more of a coronavirus, an influenza virus, a respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or a zika virus.

[0063] The inventive scheme exploits changes in end-to-end length of the attached receptor. The change in length of the Receptor corresponds in magnitude to the change in deflection / displacement distance of the MEMS cantilever. As long as the receptor curvature strength exceeds the force constant of the cantilever, this approach is independent of the physical properties of the MEMS cantilever. More specifically, the length, thickness, width, fabrication material, surface properties (Young's modulus) and attachment point do not apply in this approach. An advantage of this approach is that the fabrication process, fabrication materials, and physical characteristics 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 sensitivity of detection because these parameters change the magnitude of deflection / displacement of the MEMS cantilever. The degree of the change in sensitivity can be calculated from the physical properties of the transduction material, i.e., piezoresistors. The shorter the cantilever and the closer the Receptor attachment relative to the clamp point, the greater the sensitivity. However, since the preferred embodiment mimics a binary system with respect to deflection and the signal is experimentally measurable in the present application, sensitivity is not a parameter that needs improvement. Additionally, it depends on the relative change in the transduced value and not the absolute value, which alleviates the need to know the absolute starting deflection / displacement position of the MEMS cantilever and the specific values of the piezoresistors on MEMS cantilever. Each MEMS cantilever with the Receptor can potentially have various initial unreacted starting value, which are known prior to the introduction and challenge of the analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIGs. 1A-1D diagrammatically illustrate embodiments of abridged conformational shifting receptor for static (FIGs. 1A-1B) and dynamic (FIGs. 1C-1D) modes.

[0065] FIG. 2 illustrates exemplary sensor device configurations with variations in the number and / or relative orientation of bridged conformational shifting receptors and cantilevers.

[0066] FIG. 3 provides diagrammatic examples of a cantilever sensor according to embodiments of the inventive scheme, where panel A shows a pre-exposure condition and panels B-F respectively show conformational changes in the cantilever caused by a BCSR pulling, pushing, rotating, twisting, and combined responses.

[0067] FIG. 4 is a calculated plot of relative curvature along an organism’s genome with indicated peaks corresponding to potential Receptor candidates.

[0068] FIG. 5A diagrammatically shows the distal end of a cantilever sensor assembly, left to right, prior to exposure to an analyte, after detection, and after reset; FIG. 5B is a diagram showing device responses to analyte detection and reset.

[0069] FIG. 6A is a diagrammatic top view of an exemplary integrated sensor assembly according to an embodiment of the inventive device; FIG. 6B is a schematic showing an exemplary Wheatstone bridge as used in embodiments of the sensor; FIG. 6C illustrates a diagrammatic process flow for fabrication of a cantilever arm according to an embodiment of the inventive scheme; FIG. 6D illustrates a diagrammatic process flow for fabrication of the support base and assembly of the cantilever and support base according to an embodiment of the inventive scheme; FIG. 6E provides a diagrammatic view of the inventive integrated sensor according to an embodiment.

[0070] FIG. 7 provides a diagrammatic view of an exemplary detection system for use in detecting target analytes according to an embodiment of the inventive system.

[0071] FIGs. 8A-8B are side and top perspective views, respectively, of an optional flow cell assembly for use in an embodiment of the inventive system; FIG. 8C illustrates an exemplary approach for exposing the inventive integrated sensor to a target analyte; FIG. 8D is a diagrammatic view of an alternate embodiment for exposing the sensor to the target.

[0072] FIG. 9 is a flow diagram for a sequence of creating a cantilever-BCSR combination and using the combination for pathogen detection.

[0073] FIG. 10 is a bar graph showing a BCSR deflection when detecting a target analyte. FIG. 11 is a bar graph showing the consistent manufacturability of the BCSR sensors.

[0074] FIG. 12 is a table demonstrating the correlation between Receptor Inherent DNA Curvature to the magnitude of cantilever deflection.

[0075] FIG. 13 is a table listing details for target receptors used in a diagnostic application of the inventive system and method.

[0076] FIGs. 14A-14I provide bar graphs of test results and cantilever deflection data for nine different targets in various forms and background materials. Each figure also includes the calculated structures and predicted vs. actual system response.

[0077] DETAILED DESCRIPTION OF EMBODIMENTS

[0078] Abbreviations and Definitions

[0079] To facilitate understanding of the invention, several terms and abbreviations as used herein are defined below as follows:

[0080] As used herein, “MEMS” refers to a micro-electromechanical system incorporating micro-scale integrated devices or systems that combine mechanical and electrical components. MEMS, which are commonly fabricated using conventional integrated circuit (IC) processing techniques, e.g., photolithography, e-beam, and / or other patterning methods, can range in size on the order of 1pm to 1000pm.

[0081] As used herein, “NEMS” refers to a nano-electromechanical system, i.e., a reduced scale MEMS, which may range on the order of Inm to 1pm. MEMS consist of mechanical nanostructures, nanosensors, nanoactuators and microelectronics.

[0082] 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 commonly fabricated from silicon due to its advantageous material properties, however, a number of other materials including, but not limited to, silicon nitride (SisN4) and other ceramics, sapphire, quartz, silicon carbide, aluminum nitride, metals, polymers, or composites thereof have been .

[0083] As used herein, BCSR refers to a bridged conformational shifting receptor, which may be attached to bridge gaps between different surfaces of MEMS and / or NEMS structures, e.g., between a surface of a cantilever and a reference surface. A BCSR contact point is an area on the cantilever or on the base. The BCSR contact point may be made from a variety of materials including gold, silicon, silicon dioxide, glass, quartz, polymer, platinum, titanium, tin, aluminum, nickel, copper, and iron. The surface contact point can be functionalized with groups alcohols, alkenes, alkynes, amines, carboxylic acids, aldehydes, ketones, esters, and ethers. Modifications on the surface contact point can be different from the rest of the cantilever surface modifications for chemical, biological, and biomedical applications.

[0084] As used herein, a “Receptor” (which may interchangeably appear capitalized or all lower case) is a substance that changes conformation in response to changes in its environment. The Receptor comprises one or a plurality of molecular structures that act as binding sites for one or a plurality of analytes. Upon the binding of one or a plurality of analytes, the Receptor changes to a different conformation. A non-limiting list of receptors includes 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 repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

[0085] As used herein, a “sensor” is a device that detects or measures information from a surrounding environment and provides an output signal in response to the parameter detected or measured. A nonlimiting list of detected or measured information includes mechanical (force, pressure, velocity, acceleration, position), thermal (temperature, entropy, heat, heat flow), chemical (concentration, composition, reaction rate), radiant (electromagnetic wave intensity, phase, wavelength, polarization, reflectance, refractive index, transmittance), magnetic (field intensity, flux density, magnetic moment, permeability), and electrical (voltage, current, charge, resistance, capacitance, polarization).

[0086] As used herein, an “actuator” is a device that converts a signal into an action. It can create a force to manipulate itself, other mechanical devices, or the surrounding environment to perform some useful function.

[0087] As used herein, a “transducer” is a device that transforms one form of signal or energy into another form. The term transducer can therefore be used to include both sensors and actuators.

[0088] As used herein, a “nucleotide” is an organic molecule consisting of a nucleoside and a phosphate functional group. Nucleotides serve as monomeric units of a deoxyribonucleic acid or ribonucleic acid polymers. There are two types of nucleotides — purines and pyrimidines. The specific purines are adenine (A) and guanine (G). The specific pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA, whereas U is found in RNA.

[0089] “Amino acids” are organic compounds that contain both amino and carboxylic acid functional groups. Amino acids serve as monomeric units of peptides and proteins. Secondary, tertiary and quaternary structures influence the properties, function, and conformational dynamics of the protein.

[0090] The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.

[0091] The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0092] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of “or” is intended to include “and / or”, unless the context clearly indicates otherwise.

[0093] The following illustrative examples describe various embodiments and applications of the inventive devices and methods. These examples are not intended to be limiting. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the appended claims. Example 1 : BCSR Cantilever Configuration

[0094] The physical paradigm for the inventive system is two spring elements that are anchored at one end to a base or reference surface. The first spring is a NEMS cantilever. The second is a bridged conformational shifting receptor (BCSR), which in this example is DNA to exploit the Inherent DNA Curvature or “IDC”. The definition of a bridged conformational shifting receptor (BCSR) is any structure that changes dimensions to generate a normal force in either direction (up or down / push or pull) on the cantilever spring. In the implementation for this example, the dimensional change is orthogonal to the cantilever beam plane. The two springs are attached together near a first end of their respective physical structures, e.g., 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 support structure, i.e., a base or surface that is fixed relative to the cantilever spring. Referring to FIGs. 1A-1D, the basic concept is that when a BCSR reacts or interacts with a component within its environment it contracts by either bending (curving) or axially contracting to pull down on the lower surface of the cantilever. The complementary BCSR pairs are designed or selected to achieve the desired shape change to induce a measurable force on the attached cantilever spring sensor. FIGs 1A and 1C illustrate exemplary implementations featuring a pre-detection condition, in which the Receptor is not associated with an analyte and the cantilever is in a neutral position, e.g., “relaxed.” FIGs. IB and ID demonstrate the result of association of the BCSR with an analyte, where the BCSR contracts to pull the distal end of the cantilever toward the base.

[0095] The change in conformational shape of the cantilever can be detected by various techniques including electrical (piezoresistive, induction, capacitance, continuity (normally open or normally closed configuration), and optical (laser deflection position), quantum tunneling, fluorescence or other emission, etc.). In an exemplary embodiment, a piezoresistive transduction of the cantilever displacement / bending / deflection and a BCSR produce a change in conformation orthogonal to the cantilever beam plane after undergoing a reaction with a complementary component, corresponding to a spring contracting.

[0096] The BCSR may also expand to achieve a displacement from the neutral cantilever position. Moreover, the NEMS cantilever can be initially displaced, bent, or deflected with a BCSR base paired to its complementary nucleotide sequence to relax the BCSR or break or change a specific bond of interest. In this case, the cantilever would be restored to its unperturbed (neutral) position, resulting in a net change in displacement, binding, or deflection of the NEMS cantilever.

[0097] Displacement of the cantilever may be induced via a number of different NEMS operational modes. In the “Static Mode - System Before Reaction”, the cantilever does not exhibit significant displacement beyond the displacement arising from the combined mass of the cantilever and the BCSR(s), i.e., the total mass due to the gravitational field. In the “Static Mode - System After Reaction”, the displacement arises from small restoring force contributions from the cantilever component, where the displacement is predominately due to the conformation shape change of the BCSR. In the “Dynamic Mode”, the BCSR is modeled as a spring with an associated force constant KBCSR while in previous systems, the receptor spring has a high degree of stiffness and holds the cantilever in a permanent displaced or deflected position. Using these different operational modes, the BCSR stiffness and associated force can be dynamically assessed according to the inventive approach in which the BCSR serves as the focal point of the activity and confers a high degree of sensitivity, where its conformational changes are induced upon interacting, associating, and / or disassociating with one or multiple analytes.

[0098] Physically and mathematically, the system can be described as having multiple springs in series. In an exemplary embodiment, the device is implemented with two springs. The BCSR will apply a force on the connected force sensing cantilever spring after it reacts with its complementary pair component. The degree of reaction can yield information on the relative pair interaction. However, for preferred embodiment, the DNA will have a significant shape change and hence an associated force on the cantilever if the complementary pairs have a necessary degree of nucleotide base pair homology. This characteristic yields a binary detection scheme, i.e., if the resultant complementary pair is a significant match or interaction to yield a conformational change in dimension, it will exert a force, and if it is not a significant match or interaction, little to no force is observed and no change in conformation is observed. This is an attractive feature to ensure a high degree of specificity. Additionally, one bridged conformational shifting receptor has enough force to affect a shift in the sensing cantilever spring to be measured. This characteristic affords an extremely high degree of sensitivity.

[0099] Example 2: Static Mode Operation

[0100] For a static mode cantilever where the cantilever and BCSR(s) “springs” are in series, the force balance equation for this system is:

[0101] In a static system, the acceleration and velocity terms are zero and equation simplifies to: or where Ay = y0- y , keffy = Fext(23)

[0102] For a system with two serial springs, the effective spring constant is the sum to the two springs, where the effective spring constant keff=kc+ kBCSSR. (24)

[0103] For a more general system description of n springs (a cantilever with n BCSRs), the effective spring constant would be:

[0104] The external force Fext is

[0105] Fext=Fc+ FBCSR(26)

[0106] Equation (23) then becomes where the force exerted by the cantilever is Fc= kcy, and the force exerted by the bridged conformational shift shifting receptor is FBCSR= kBCSRy, where KBCSR, is the force constant of the bridged conformational shape shifting receptor.

[0107] For a system of springs in series, the forces from both springs are different. Since both springs are connected at the common point at the distal end of the NEMS cantilever, the displacement on both springs must be equal, i.e., yc=yBcssR (28)

[0108] The displacement of the BCSR can now be evaluated before and after reaction with the complementary pair.

[0109] It important to note Equation (28), which is the master equation, mathematically demonstrates that there is no dependance on the characteristics of the NEMS cantilever, i.e., spring constant, length, width, thickness, Young’s modulus and / or mass loading. It is dependent only on the dimensional change of the BCSR which results in change in the NEMS cantilever displacement / deflection / bend which is equal in magnitude.

[0110] Static Mode - System Before Reaction

[0111] Prior to reaction with the complementary and the attached BCSR (ssDNA or RNA), the boundary conditions are such that the FBCSRb= 0, by adjusting the gap between the cantilever tip and stationary base. At this stage both ends of the unreacted BCSR are attached and adjusted so the is no slack or just stretched taught enough to cause a slight displacement. The slight displacement would provide a position control to ensure unreacted BCSR attachment. Therefore, the system at equilibrium, FTb= Fcbefore the BCSR has reacted. The displacement for the cantilever spring is due to the mass of the cantilever and the mass of the BCSR(s) or the total mass due to the gravitational field, mT= mc+ mBCSSR(29)

[0112] The total mass for the NEMS cantilever with the attached BCSR(s) is essentially zero, mT« 0. The approximate mass of a typical NEMS cantilever is on the order of tens of femtograms (~10‘14g). The approximate mass of a typical large BCSR, e.g., DNA is on the order to tens of zeptograms (~10'2° g). Additionally, the cantilever spring constant is much larger in magnitude relative to the gravitational force constant. Therefore, the cantilever does not show significant displacement.

[0113] Thus, before reaction of the BCSR, the displacement of the NEMS cantilever is yBcssRb= yCb« 0, i.e., no displacement prior to reaction with BCSR’s complementary pair. If there is any measurable displacement it can be recorded as the baseline representing the starting displacement position.

[0114] Static Mode - System After Reaction

[0115] After introduction of the complementary pair, the cantilever spring measures the amount of apparent or effective displacement. If the force of the of the BCSR is large relative to the restoring force of the cantilever, the displacement of the cantilever in either direction is due to the displacement of the of the BCSR. Thus, at equilibrium, Ayc= yBcsRa- Thecantilever measures the apparent or effective displacement where the total exerted force on the connection point at the distal end of the NEMS cantilever is Fext= pBcsRa+ Re¬

[0116] If pBcsRa» Pc, the total force equals, Fext= FBCSRa. The displacement under these conditions has very small restoring force contributions from the cantilever component and the displacement is predominately due to the displacement caused by the confirmation shape change of the BCSR. Some embodiments utilize a NEMS cantilever with a spatial coordinates definition in Euclidian space. The cantilever displacement in such a system will be defined orthoganally to the cantilever beam in the y direction. The gap between the attachment point(s) on the cantilever and the BCSR attachment base is on the order of nanometer. The 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 a Young’s modulus, E, moment of inertia, I, and a force, FBCSR, exerted by the bridged conformational shift shifting receptor. where the moment of inertia 33)

[0117] Substituting Equation (33) into the cantilever displacement Equation (32) and rearranging yields the force constant, kc.

[0118] Thus, a desired cantilever stiffness can be attained through selection of cantilever dimensions ( / ., H, and W) and fabrication materials (E).

[0119] BCSRs can be attached at different points along the cantilever. The BCSR attached at the tip of the distal end provides the maximum displacement. As the BCSR attached at the tip ends is compressed to its maximum, further compression of the cantilever by the other BCSRs can also be achieved. The complementary configuration of the BCSR, i.e., its compressed form, is achieved through its attachment to the cantilever. The details of this attachment are discussed as specific embodiments to provide a clear explanation, however, it is not intended to limit to the following specific discussion.

[0120] It should be noted that the cantilever’s geometric design characteristics and fabrication materials play a small role in the displacement of the cantilever. Rather, the key is the BCSR, which determines the displacement vector along the line between the two attachment points. This vector need not be precisely orthogonal, i.e., 90°, to the cantilever beam, however, if off-orthogonal configurations occur, reduced displacements can be expected. If the reacted BCSRa structure, i.e., the “spring”, is stiff and relatively uncompressible, and if multiple different BCSRs having different attachment geometries are combined on a cantilever, the displacement may not exceed the length of the most orthogonally-attached BCSR. Where multiple BCSRs are to be attached to the cantilever, the preferential physical attachment on the tip may be achieved by physically displacing the tip towards the base attachment plate and so that the shortest distance in the gap will bind / attach the BCSRain an orthogonal orientation at the cantilever tip. The BCSRa cannot be attached to other areas along the length of the cantilever due to the physical length constraints of the BCSRa. Attaching the BCSRa, ensure a specific and optimal attachment geometry (orthogonal) and attachment location (cantilever tip). After attachment of the BCSRa the complementary pair is released or restored to the unreacted state to achieve a different conformational structure of the BCSRb. The system is then ready for use for detection by a reaction with its complementary pair to form the BCSRa.

[0121] The NEMS cantilever can be calibrated for a specific signal transduction method. 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-calibrating for thermal drift so that all resistors are in close proximity and, thus, in thermal equilibrium. The potential across the Wheatstone bridge is calibrated for the specific resistor values and piezoresistive response as a function of displacement of the cantilever to determine the response slope,

[0122] Ay’or(v35)7

[0123] By measuring the potential change, the magnitude of the displacement can be obtained. However, in embodiments employing DNA or RNA, the displacement value of any magnitude is needed to confirm a match in the complementary pair, the analyte. Once a potential is detected, a significant match or interaction can be confirmed.

[0124] Example 3 : Dynamic Mode Operation

[0125] The dynamic mode mathematic model can be derived for a spring attached to a NEMS cantilever where the BCSR is attached to a stationary structure. Here it is assumed that the BCSR in somewhat flexible and can be modeled as a spring with an associated force constant kBCSSR. In the static mode operation, the receptor spring is extremely stiff and holds the cantilever in a permanent displaced or deflected position. The BCSR stiffness and associated force can be dynamically assessed. The equation of motion for this system is as follows: where

[0126] Substituting keqinto Equation (37), yields

[0127] Solving Equation (39) to determine the resonance frequency, yields

[0128] If the resonance frequency is determined and the dimensional characteristics of the NEMS cantilever, mass, and the Young’s modulus are known, then the bridged shape shifting receptor spring constant can be calculated (Equation (40)).

[0129] Example 4: Cantilever Configurations

[0130] Different embodiments of cantilever configurations can be formed using one or a combination of integrated sensors where the integrated sensors are configured to form a bridged cantilever geometry. As used herein, an “integrated sensor” refers to a cantilever with means for measurement of cantilever deflection relative to a support base with a fixed gap that is bridged by a Receptor. Exemplary configurations of a device for detecting an analyte in a sample are diagrammatically illustrated in FIGs. 1A-1D. Note that while a single cantilever is shown, multiple cantilevers may be arranged in an array. The device comprises one or a plurality of integrated sensors and a detector. FIGs. 1A and 1C each illustrate a neutral or physically analyte-disassociated configuration when the Receptor is not associated with the analyte. The integrated sensor 2 is configured to form a bridged cantilever geometry including a cantilever 10 with a first surface 12, a second surface 14 on a base 16 (or other stable surface), and a Receptor 20 with a receptor recognition site 22. Each of the first surface 12 and the second surface 14 include one or more contact points for attachment of one or more Receptors 20. FIGs. IB and ID illustrate the devices of FIGs. 1A and 1C, respectively, showing a physically analyte-associated configuration when the Receptor is associated with the analyte. The first end of the Receptor is attached to the contact point on the first surface and the second end of the Receptor is attached to the contact point on the second surface. One or more analytes are associated with the Receptor through one or more molecular structures on the Receptor. Due to the conformational change of the Receptor in the presence of one or a plurality of binding analytes, the first end and the second end of the Receptor have changed from its resting state. The relative distance and angle between the first end and the second end of the receptor are different with and without the binding of analytes.

[0131] Example 5: Other BCSR Cantilever Configurations

[0132] Neutral, or physically analyte-disassociated configurations of different sensors are shown in FIG. 2, panels A-E, where no receptor has been associated with an analyte. These non-limiting examples show a variety of possible combinations of BCSRs and cantilevers. Panel A shows a single bridged receptor between a stationary surface and a parallel cantilever. Panel B illustrates a possible implementation of multiple bridged receptors between a surface and a parallel cantilever. Panel C shows an exemplary arrangement having a single bridged receptor between a surface and two parallel cantilevers. Panel D shows an exemplary device having a single bridged receptor between two fixed surfaces whereby analyte association induces stress in the suspended ‘cantilever’ material. Panel E illustrates a sensor configuration having a single bridged receptor between a surface and a cantilever in a planar orientation. Based on the disclosure herein, a skilled artisan would be able to devise a variety of different cantilever-receptors combinations to provide a sensor. .

[0133] Example 6: BCSR Receptors

[0134] Polynucleotides, polypeptides, and polysaccharides may all change conformation in response to changes in their environment, such as binding to substrate or analytes, changing salt concentrations or pH, phosphorylation, etc. In addition to these biopolymers, other materials are also known to change conformation in response to changes in their environment.

[0135] In some embodiments, the Receptor comprises an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

[0136] The Receptor comprises one or a plurality of molecular structures that act as binding sites for one or more analyte. Upon the binding of one or more analyte, the Receptor changes to a different conformation by shrinking, bending or otherwise modifying the gap that is bridged by the BCSR.

[0137] The information contained within an organism’s genetic material far exceeds the simple nucleotide coding sequence. Certain patterned organization of specific nucleotides induce a higher order conformation or curvature of the genetic material. These structures serve critical biological functions such as controlling gene expression, chromosome packaging, transcription initiation and termination, recombination, DNA replication, and nucleosome positioning.

[0138] One known example is the variation of the DNA double helix structures with inherent DNA curvatures. While single-stranded DNA (ssDNA) exhibits no inherent curvature, the hydrogen bonds between adenine and thymine nucleobases are not perpendicular to the helical axis, such that inherent deflection of the axis can be induced upon binding with certain analytes with modification of composition, position, and length of certain nucleotide sequences. For example, the end-to-end distance of an 85nm doublestranded DNA (dsDNA) helix can be reduced to 65nm or more.

[0139] FIG. 4 provides a sample plot of sequence-dependent spatial trajectory of a DNA double helix and the distribution of inherent curvature along the DNA molecule. Typically, the nearest-neighbor wedge model is implemented to calculate the overall DNA structure using local helix parameters: dinucleotide twist, tilt, and roll angles. FIG. 4 shows two peak curvature regions at approximately 4,600 and 26,000 along an adenovirus genome, indicated by the arrows.

[0140] In some embodiments, the Receptor comprises a nucleic acid, a nucleic acid analogue, a double-stranded DNA (dsDNA), a dsDNA analog, a single-stranded DNA (ssDNA), a ssDNA analog, or a peptide nucleic acid.

[0141] 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.

[0142] In some embodiments, the Receptor is 50 - l,000nm in length. A skilled artisan would be able to design / produce the length of the Receptor to optimize the performance for a specific application.

[0143] Another known example of conformation variations is the two conformation isoforms of the prion protein. The cellular isoform of the prion protein is the alpha-helix rich prion protein (PrPC), while the prion isoform is a beta-structure-rich insoluble conformer (PrPSc).

[0144] Example 7: BCSR Response to Analytes

[0145] Analytes can be associated with Receptors on the cantilever and trigger conformational changes of the receptors. Analytes and Receptors are held together through non-covalent interactions, including electrostatic, Van Der Waal’s, 7t-effects, hydrophobic and hydrophilic interactions.

[0146] In some embodiments, 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 repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

[0147] In other embodiments, the analyte comprises a complementary nucleic acid, a complementary nucleic acid analogue, a complementary dsDNA, a complementary dsDNA analog, a complementary ssDNA, a complementary ssDNA analog, a complementary RNA, or a complementary RNA analog.

[0148] The methods and devices described herein are not limited to the use of any particular sample type. Examples of samples that may be tested using the inventive devices and method include lachrymal fluid, saliva, buccal fluid, a bronchoalveolar lavage fluid, mucus, a nasal sample, a nasopharyngeal fluid, urine, fecal, tissue, blood, plasma, serum, cell culture, bodily fluid, tissue biopsy, apocrine fluid e, or eccrine fluid. Types and sources of samples may further include forensic, aerosol, soil, water, food, ingredients, raw material, in-process, byproducts, final products, or quality control samples.

[0149] In some embodiments, the sample may be a virus, bacterium, phage, yeast, mycoplasma, fungus, human cell, animal cell, plant cell, insect cell, or any combination thereof. The inventive approach has no molecular limitations on its ability to detect any virus (bacteria, fungi, etc.) regardless of nucleic acid, protein, or other composition.

[0150] The association of the BCSR with the analyte can induce various conformational changes causing a change in the relative position of the cantilever and reference. FIG. 3, panels A-F, diagrammatically illustrate relative movements of an exemplary sensor with a single bridged conformational shifting receptor between a surface and a parallel cantilever. FIG. 3, panel A shows the exemplary sensor with a single bridged conformational shifting receptor in a physically analyte-disassociated, i.e., neutral, configuration. FIG. 3, panel B shows the exemplary sensor with a single bridged conformational shifting receptor is in a physically analyte-associated configuration showing a pulling action on the cantilever. FIG. 3, panel C shows an exemplary sensor with a single bridged conformational shifting receptor in a physically analyte-associated configuration showing a pushing action on the cantilever. FIG. 3, panel D shows the exemplary sensor with a single bridged conformational shifting receptor is in a physically analyte-associated configuration showing a rotating action on the cantilever. FIG. 3, panel E shows an exemplary sensor with a single bridged conformational shifting receptor in a physically analyte-associated configuration showing a twisting action on the cantilever. FIG. 3, panel F shows an exemplary sensor with a single bridged conformational shifting receptor in a physically analyte-associated configuration showing a combined pulling and twisting action on the cantilever. A wide variety of permutations are possible beyond the illustrated examples. To provide another alternative configuration, the planar orientation shown in FIG. 2, panel E can be modified so that the BCSR causes relative pulling, pushing, rotation and / or twisting between the cantilever and the support surface. A skilled artisan would be able to vary the configuration of the receptor with respect to a specific application without undue experimentation.

[0151] In most embodiment, the interaction, association, or disassociation of the Receptor with one or a plurality of analytes induces a change in the relative positions 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 surface and the second surface is 1 — 1,000 nm.

[0152] Example 8: Resettable BCSR

[0153] Referring to FIG. 5A, association of the BCSR 52 with a target analyte 54 may cause inherent curvature or axial contraction of the bridged Receptor 52, deflection of a piezoresistive-coupled cantilever 50 (shown in step 2), and thus a change in resistance. In some embodiments, the device that is used once and then discarded after step 2. Other embodiments may be reset by any means of disassociating the Receptor 52 and analyte 54, including thermal, electrical, electric chemical, or chemical methods. The analyte may be disrupted, destroyed, or removed by flushing or evacuating the test chamber. In some embodiments, a relatively alkaline buffered solution is flushed through the test chamber to denature a dinucleotide complex and separate the analyte from the Receptor, e.g., step 3. Disassociation of the analyte from the BCSR causes the Receptor to return to its original conformation, relaxing the piezoresistive-coupled cantilever, and thus causing a change in resistance as shown in FIG. 5B.

[0154] Example 9: Signal Transduction

[0155] The function of piezoresistive materials is well known in the art. Briefly, in piezoresistive materials, a change in a physical property is converted into a change in an electrical physical property. In the context of the present cantilever sensors, a flexible piezoresistive material is bonded to or coated on a substrate surface that is itself configured to deform upon application of a force to the material. Upon bending of the substrate surface, the crystalline structure of the piezoresistive material is also subjected to deformation or stress, thereby altering its resistive properties. The sensitivity coefficient is determined as the ratio of change in stress to change in resistance (i.e., Stress / Resistance). It is important to note that the transduction material comprises a piezoresistor coating on the substrate surface, and not the substrate surface itself.

[0156] The deflection / displacement of the first surface and / or the second surface can be measured by placing a transducer element in direct contact with either the first or second surface. Signal transduction, via a transducer measures the change in a physical property as it relates to a change in an electrical physical property (V, I, R) change, electromagnetic amplitude magnitude change, EM field strength change, EM frequency change, EM position vector change, etc. The magnitude 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 crystalline structure of the piezoresistive material. The deformation or stress of the crystalline structure changes the resistive properties. The sensitivity coefficient is change in stress divided by the change in resistance (which is a slope. 8Displacement / 8Resistance or 8D / 8R).

[0157] The transduction material is a patterned piezoresistor coating on the surface. Mathematically, A displacement = (8D / 8R) A resistance or equally, A resistance = (8R / 8D) A displacement.

[0158] Referring to FIG. 6A, a MEMS cantilever-based sensor assembly 60 is diagrammatically illustrated. Cantilever arm 62 is formed from 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 to the BCSR(s). The proximal end of arm 62 is bonded to a support base 64, with electronic circuitry patterned thereon. It should be noted that the interconnect pattern as illustrated is representative only and does not depict a layout of electrical connections in the device, (c.f. Example 7 below, which describes the Wheatstone bridge circuitry that is used for measurement of resistance changes.) Electrical conductors 66 extend at least partially along the length of cantilever arm 62, embedded or otherwise connected to the piezoresistive material in the cantilever to communicate signals generated within the piezoresistive material to the various conductors and bond pads 68 on support base 64 to provide electrical signals (e.g., Vin, Vout, Vcc) to and from external devices as will be described further below. The base portion 64 will typically be bonded to a printed circuit board (not shown) with conventional wire bonding techniques used to create connections. The base portion 64 and PCB may be sealed within a package or epoxy sealant for protection and to facilitate connection to a socket or multi-pin connector. As previously described, in most embodiments, the BCSR is typically attached to the underside of the distal end of cantilever arm 62 and the upper surface of the support base 64 to bridge the gap.

[0159] In some embodiments, fabrication of the MEMS piezoresistor cantilever assembly employs conventional semiconductor fabrication techniques, which may include standard photolithographic patterning steps, chemical and plasma etching, e-beam writing, thermal diffusion and / or ion implant for doping, chemical vapor deposition, sputtering, etc.

[0160] FIG. 6C diagrammatically illustrates an exemplary processing sequence for fabricating the cantilever arm on a silicon on insulator (SOI) wafer according to an embodiment. Based on this exemplary sequence, it will be readily apparent to those of skill in the art that variations in the process sequence may be made without deviating from the general principles of the inventive approach, which is to provide a precision cantilever with repeatable dimensions and performance.

[0161] The starting substrate is silicon on insulator (SOI) wafer, i.e., silicon and oxide (SiO?) on a handle layer. Exemplary starting wafer layer thicknesses may be on the order of 10 pm, 1 m, and 400pm respectively. The following steps for fabricating a cantilever structure according to an embodiment of the inventive approach are labeled as (a) through (m) in FIG. 6C. The various steps utilize conventional semiconductor processing techniques as are known in the art and are therefore not described in detail.

[0162] (a) a thermal oxide (SiO?) (~ 500nm) is grown on the wafer.

[0163] (b) after patterning of the structures, the oxide is etched to expose the handle layer.

[0164] (c) N-type doping and diffusion in exposed structures.

[0165] (d) remove surface oxide.

[0166] (e) thermal oxidation (~100nm).

[0167] (f) pattern and oxide etch to open structures to define the piezoresistors in the Si layer followed by P-type doping / diffusion. (Ion implant may also be used for this step.) An exemplary geometry for the piezoresistors is approximately 10pm wide x 50pm long (2x in series), with a diffusion depth of about 0.25 - 0.5pm, estimated depending on the doping concentration.

[0168] (g) oxide etch and grow thermal oxide (~100nm).

[0169] (h) pattern and etch oxide to expose contact windows.

[0170] (i) deposit contact metallization (Al / l%Si) and etch to pattern.

[0171] (j) deposit SisN4 (“SiN”)passivation using plasma enhanced chemical vapor deposition (PECVD), pattern and etch.

[0172] (k) pattern gold or Ti / Au onto cantilever tip for BCSR attachment.

[0173] (l) etch the cantilever and dicing streets (cf. steps (s) and (t) below).

[0174] (m) etch handle wafer to release devices (cf. step (u) below).

[0175] The resulting devices correspond to the cantilever arm, i.e., one of the two surfaces to which one end of the BCSR(s) will be attached. Exemplary dimensions for some embodiments of the cantilever will be on the order of 400 - 450pm in length, 100 - 115pm in width and about 3.8 - 5pm thick. Resonance of the cantilever will be on the order of 15- 40 kHz. This cantilever will be permanently attached to an elevated pedestal which is orthogonally attached to a rigid surface, i.e., a mounting base, which forms the second component of the sensor device. Fabrication of the mounting base is described below.

[0176] FIG. 6D diagrammatically illustrates the process flow for fabrication of the second component of the sensor, i.e., the mounting base, and assembly of the cantilever structure to the base. The base provides electrical connection between the piezoresistor elements on the cantilever structure to external interconnect. The following steps for fabricating the base structure according to an embodiment of the inventive approach are labeled as (n) through (u) in FIG. 6D. The following steps utilize conventional semiconductor processing techniques as are known in the art and are therefore not described in detail.

[0177] (n) starting with a SOI wafer, a layer of thermal oxide is grown on the Si surface.

[0178] (o) polysilicon is deposited, e.g., PECVD, on the oxide surface and patterned to define the pedestal for supporting the cantilever.

[0179] (p) the oxide is patterned and etched to form an opening for connection to a substrate ground.

[0180] (q) Ti / Au is deposited on top of the wafer and patterned to define electrical contacts and the gold (or other material) area for BCSR attachment . (r) the upper surface of the cantilever on the handle wafer is positioned relative to the base wafer to align the proximal end of the cantilever with the polySi pedestal and the gold area on the distal end of the cantilever with the gold area on the base. (Alignment guides will have been patterned on both wafers in accordance with standard semiconductor processing techniques.) Once aligned, the assembly is subjected to thermal processing for gold-silicon eutectic formation. As is known, Au-Si eutectic formation occurs at around 360-370°C. Exemplary temperatures for forming the bond will be on the order of 410- 470°C.

[0181] (s) etch the cantilever and dicing streets.

[0182] (t) carrier attachment and wafer thinback to release the handle layer from cantilever.

[0183] (u) final release for completed device.

[0184] Bond pads formed in step (q) are electrically connected to corresponding connectors on the PCB to provide external connections.

[0185] Keys steps within the process include steps (o), (q), and (r), the combination of which allow for formation of a repeatable and well-defined gap between the bottom surface of the cantilever and the upper surface of the base. Specifically, the gap between the contact area near the distal end of the cantilever (labeled “Gold contact #1” in FIG. 6D) and the gold contact area directly below the distal end of the cantilever (labeled “Gold contact #2” in the figure) is a key structural component of the inventive sensor device. These gold contact surfaces may be within a range of about O.lnm2, e.g., the approximate diameter of a single receptor molecule, to l,000nm2or larger. The resulting structure defines a gold- plated / coated gap between two surfaces which serve as attachment points for the molecular moiety attachment ends. As previously noted, while gold is used in the described examples as the material for the BCSR contact point, the contact area material may be a variety of other materials including silicon, silicon dioxide, glass, quartz, polymer, platinum, titanium, tin, aluminum, nickel, copper, and iron. The base surface is a rigid structure that will not bend or become distorted, while the cantilever surface is flexible and capable of bending or deflecting. This physical configuration is the NEMS device that serves as the detection unit. The height of the polysilicon pedestal formed in step (o) can be controlled to a tight tolerance within a few nanometers using known deposition techniques. The selected pedestal thickness, combined with the thickness of the gold contact (step (q)) and eutectic bond formed in step (r), which can be expected to be very thin (on the order of 200 A or less) defines the gap. The gap can vary from 5nm to hundreds of microns, primarily by controlling the thicknesses defined in steps (o), (q), and (r). This integrated cantilever structure provides the foundation to be further modified and adapted to attach / bond a single or multiple molecular moiety(s) to bridge the gap.

[0186] FIG. 6E provides a diagrammatic view of the inventive integrated sensor 100 according to an embodiment fabricated according to the process flows shown in FIGs. 6C and 6D. (It should be noted that this figure is not to scale. Rather, the dimensions are highly exaggerated to facilitate illustration of the key components of the inventive integrated sensor, including the Receptor placement in the gap.) The Legend provides a key for the materials used in fabrication of sensor 100.

[0187] This ability of the inventive approach to provide precise and repeatable control over the gap dimension overcomes the shortcomings of prior efforts to fabricate reliable cantilever sensing devices for use for bridged sensing applications.

[0188] In some embodiments, the ends of a Receptor comprising ssDNA is attached or chemically bonded to the two gold contact areas using a thio-gold chemistry. Other attachment chemistries are available depending on the molecular structure of the bridge moiety. This chemistry has been proven to possess the necessary physical strength to remain attached during a molecular structural shape change. The NEMS integrated cantilever unit is designed and fabricated to a relatively weak desired force constant compared to the force applied by the structural in the molecular moiety. This configuration provides the necessary condition for the cantilever surface to be displaced during the change in shape configuration of the molecular moiety, e.g., dsDNA. When single strand (ssDNA) is attached at each end between the NEMS cantilever and the fixed surface to the integrated NEMS cantilever it is relatively linear and does not apply much force to bend or displace the flexible cantilever structure for in normal or neutral position, i.e., flat. When highly complementary or matching ssDNA binds / reacts to the attached ssDNA element, it forms the corresponding dsDNA helical structure. The dsDNA molecule is specifically selected via modeling and experimental validation to possess a highly curved structure. The newly formed curved structure possesses a force constant much greater than the force constant of the MEMS cantilever and causes it to bend or deflect. The displacement is measured by the attached piezoresistor(s) placed on or integrated into the surface of the NEMS cantilever. The piezoresistor is positioned onto the cantilever at a location that will achieve the greatest degree of deformation, i.e., near the pedestal mount, via a Wheatstone bridge configuration. The deformation of the piezoresistor material causes a change in the crystalline structure and thereby inducing a change in the resistance value that is measured by a change in the potential across the Wheatstone bridge design.

[0189] Example 10: Detection System

[0190] FIG. 7 diagrammatically illustrates a prototype detection system 70 used in preliminary testing and evaluation of the inventive approach for detecting analytes using the integrated sensor. This embodiment operates using the principles of a Wheatstone bridge, which is two simple series-parallel arrangements of resistances connected between a voltage supply terminal, where the resistances are the piezoresistive coating on the cantilever arm 62 which are converted into an analog signal.

[0191] Voltage source 72 provides voltage across the Wheatstone bridge that is defined by PCB 64 and arm 62. FIG. 6B provides a diagram of a half-active, full Wheatstone bridge that may be employed in some embodiments. Sensor assembly 60 may be plugged into a connector socket 63 that provides connection to voltage source 72 and pre-amplifier 74 that receives the signal from sensor assembly 60 for measuring the voltage change from the Wheatstone bridge. The measured voltage change is converted from analog to digital at A-to-D converter (ADC) 76. Preamplifier 74 provided a 100-gain amplification signal from the Wheatstone bridge to the ADC which was then input into the system computer controller 78 which receives measurement signals as well as providing control signals for operation of the system 70, including control, manipulation, and operation of the optional flow cell assembly 80, which is further described below with reference to FIGs. 8A-8B. The computer controller displays the data graphically on the screen as function of cantilever output voltage as a function of time. The software was developed using NI-DAQmx (version 21.3). The amplified output signal was 20 nm / volt.

[0192] Among the flow cell controls that may be provided by computer controller 78 are XYZ directional controls for precision multi-axis positioning stage 79 which is used to position the sensor assembly within a test well or chamber, flow parameters, and valves for introducing analyte and / or flushing / rinsing the flow cells. Computer controller 78 may also include memory for storing results and links to external memory and / or communication systems for reporting measurement results.

[0193] A magnified optical imaging system was designed, fabricated, and tested to visually observe the cantilever positioning over the slide base and flexure of the MEMS cantilever. A camera captured the image / video of the magnified cantilever image and projected onto a computer screen and / or transmitted the visual information to a computer. In a test set-up, positioning stage 79 was a piezo flexure stage mounted beneath the cantilever to adjust the X, Y, and Z position of the gold coated surface from which the Receptor bridges to the cantilever tip. Piezo flexure driver unit controlled the exact positioning of the stage to enable nanometer-scale control of the gap distance between gold surfaces. To achieve a cost effective and deflection sensitive cantilever system, a piezo flexure stage was selected with an open loop with a 267 nm / V of X, Y, and Z moment component. For every volt applied to each channel the piezo flexure element moved 267 nm, for a full length of a 20 pm travel range (@ 75 volts max). The piezo flexure stage as well as all the other dynamic movement components were mounted onto a 18” x 24”, breadboard optical bench with vibration isolation legs. A slide holding attachment fixture was mounted directly onto the piezo flexure stage. A gold coated glass slide was attached to the slide holder attachment to provide a base bioreceptor attachment area. To adjust the gap between the cantilever and base, the piezo flexure stage was raised or lowered as required during the various experimental processes. The piezo flexure stage may be manipulated manually with a control knob on the piezo flexure control module or via a computer software program and GUI running on the computer connect through a USB cable to both ports. The cantilever mounting fixture consisted of multiple components, an elevated mounting fixed stage, and magnetic cantilever mounting head for easy removal and replacement. The cantilever mounting head allowed the PCB of the MEMS cantilever to be fastened to it via a screw adjustable clamping device. Additionally, a microscope video camera was mounted on the optical breadboard to visually inspect the MEMS cantilever alignment and bending during the execution of the experiments.

[0194] In an experimental set-up, the amplifier circuit supplied 2.048 volts across the Wheatstone bridge. The circuit provided a positive potential difference when the MEMS cantilever was deflected upwards and a negative potential difference when bent or pulled downward. Two of the piezo resistors were placed a base of the cantilever, while the other two piezo resistors were placed near the base of the cantilever on the stationary section of the MEMS device. The two piezo resistors were placed on the bottom portion of the cantilever to experience a change in physical dimensions of expansion (upward bend) and compression (downward bend). This physical change in the piezoresistive material on the cantilever provided the transduction means to detect the extent of bending in the MEMS cantilever. Connector socket 63 allowed rapid replacement of the sensor assembly. To provide a positive control from the MEMS cantilever to computer output display, the stage was maneuvered upward with the vernier control knobs (rough first then fine) until the gold coated glass slide just touched the MEMS cantilever tip. The MEMS cantilever voltage output was monitored as the tip was about to become engaged and adjusted to a baseline offset voltage which represented a zero deflection. The mounted MEMS cantilever was then deflected upward by moving the piezo flexure stage upward and measuring the voltage chance on the computer data output window. The MEMS cantilever voltage change was monitored to check on the appropriate deflection change on the upward and downward piezo flexure controller sweep, i.e., voltage magnitude and voltage direction (+ / -). This process ensured that the MEMS cantilever and piezo flex stage responded in the desired fashion before the Receptor was attached and the detection experiment was executed.

[0195] Preliminary tests included bending the cantilever both up and down with the stage controller to examine the data acquisition (DAQ) output voltage. The stage was positioned beneath the fixed cantilever using the vernier dials to a position where the cantilever was visually observed to engage the slide, followed by slightly backing stage off to disengage the cantilever. Next the Z direction was adjusted upward via the piezo flexure control system to reengage the cantilever and to bend it over a range of values. The camera mounted above the cantilever recorded the original cantilever position before bending. As the cantilever tip position was displaced, it came in and out of focus. Bending the cantilever down was accomplished by adjusting the cantilever tip beneath a suspended microscope cover slip fixed to the stage. As the cover slip was lowered on top of the cantilever it forced the tip in a downward direction. The applied stage controller voltage versus the DAQ response in voltage and conversion to nanometers (IV =10 nm) was then plotted. As the cantilever was bent upward, the DAQ voltage increased. Conversely, as the cantilever was bent downward, the DAQ voltage decreased. This was more analogous to the response expected when the bridged Receptor IDC curvature would force the cantilever down toward the stage.

[0196] Example 11 : Optional Flow Cell Assembly

[0197] Referring to FIGs. 8A-8B, flow cell assembly 80 provides an optional approach for precisely and repeatably positioning cantilever-based sensor assembly within a reaction chamber or flow cell whereby analytes are able to interact with the Receptor. Flow cell assembly 80 has a base plate 85 with a laminated structure having capillary channels 87 extending between the layers. The channels 87 provide fluid connection between connectors 82 and 84, which extend from the upper surface of the plate 85, and test well 86. Connectors 82 and 84 are configured to mate with standard laboratory tubing connectors for feeding fluids into test well 86. Connectors 82 provide in and out flow of a fluid for flushing the test well and sensor system. The fluid used for flushing may be a sterilizing solution, or it may be solution selected to reset the sensor, e.g., alkaline buffered solution, to dissociate the receptor and analyte. Connector 84 provides for introduction of an analyte fluid into the test well via a pipette, dropper, or other applicator.

[0198] FIG. 9 is a flow chart illustrating the steps of an exemplary pathogen detection process using the system of FIG. 7 including the optional flow cell assembly In step 91, the BCSR is produced using double-stranded semi-modified DNA bio-receptor, in which one strand is modified at both 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 floor of test well 86. (Note that when using integrated sensor 100 (FIG. 6E), the BCSR bridge will span the gap between the cantilever tip and the support base.) In step 92, the flow cell assembly is raised by the computer controller providing commands to positioning stage 79 to create a 150nm gap between the cantilever tip and the test well floor. Next, the dsDNA BCSR is attached to the cantilever tip and the well floor in step 93. In step 94, the positioning stage is controlled to lower the flow cell assembly by approximately 5nm to induce a cantilever deflection. Note that the exact distance is not critical. This initial cantilever deflection is used to confirm successful formation of the initial bridge and for a system diagnostic to confirm bridge integrity during testing Next, the dsDNA is denatured in step 95 by introducing a chaotropic agent or other denaturing fluid through tubing 88 and flow channel connectors 82. This leaves the modified strand permanently attached across the gap between the cantilever tip and the well floor. The unmodified receptor complement is then flushed out of the test well in step 96. In this sequence, steps 91-96 can be grouped into a sequence known as “Bio-Bridge Construction.”

[0199] The following steps make up the “Pathogen Detection” sequence. In step 97, the sample containing a suspected receptor complement (e.g., the pathogen for which testing is being performed) is introduced into the test well through sample port 84. In step 98, if the sample includes the suspected receptor complement, the dsDNA will contract or bend as the result of IDC, causing the cantilever to be pulled down. The deflection of the cantilever will cause the piezoresistive element to change resistance, which is detected by the detection assembly and communicated to the computer controller for output of the test result, i.e., a positive test, and storage in the computer memory or external memory. If the sample does not include the suspected receptor complement, no deflection will occur, and a negative result will be reported by the computer controller.

[0200] Referring to FIG. 8C, exposure of the integrated sensor 100 to the analyte for pathogen detection can be achieved by any of a number of different methods, including, for example, depositing sensor 100 in and / or affixing it to the bottom of a test well 102. Test well 102 may be a single container or may be one of an array of wells in a multi -well plate, such as those used in high-throughput screening of samples. Test well 102 may be filled with a sample processing reagent into which analyte samples would be introduced. It should also be noted that there is no limit as to the size or dimensions of a reaction chamber or test well and that any number of integrated sensor assemblies 100 may be placed in the reaction chamber.

[0201] Electrical connectors 110 provide connection between the conductor pads on (each) sensor 100 and the system electronics as described with reference to FIG. 7. Although not shown, the PCB to which the integrated sensor’s bond pads are connected, e.g., via die attach, ball wedge bonds, wire bonds, or similar, may be connected to a multi-contact socket, a multi-pin plug, a flex cable pinout, or similar structure to facilitate rapid electrical connection of the sensor to and disconnection from the system electronics. Analyte 106 can be introduced into test well 102 by a dropper 104, pipette, or similar applicator. In some applications, the test well, rather than being a container configured for retaining a liquid, may be a chamber, canister, or cuvette 120 into which an aerosol sample 122 can be introduced to expose the sensor 100, as shown in the example of FIG. 8D. (Note that electrical links to the external devices are not shown.) The aerosol sample can be introduced using an approach similar to that used in a breathalyzer. While the underlying measurement principles are different for detecting alcohol (via oxidation of ethanol to acetic acid) using a conventional breathalyzer and detecting pathogens or other analyte via interaction with the Receptor of the inventive integrated sensor, the exposure mechanism is similar to the extent that a subject may be able to simply expel air 122 into a mouthpiece 124 connected to the test chamber 120 to allow direct detection of the presence of the Target by the sensor 100.

[0202] Association between the Target analyte within the sample and the bio-bridge Receptor induces a structural change in the bridged molecule (BCSR) which causes the cantilever to deflect. Upon deflection, the piezoresistive elements in the cantilever induce a change in output voltage to generate a detection signal. The test well may be of any shape or dimensions, and there is no limit to the number of bio-bridged cantilevers that can be employed for testing. Alternative structures and approaches for exposing sensor 100 to the analyte to be tested will be readily apparent to those of skill in the art.

[0203] Example 12: BCSR Sample Processing Reagent (SPR)

[0204] A sample processing reagent (SPR) for detecting an analyte in a sample is provided herewith. The SPR comprises a buffer, an enzymatic activity inhibitor, a denaturant, a low ionic strength detergent, and an oxidizing agent. The SPR enables the receptor in the integrated sensor to associate with one or a plurality of analytes in a sample. The SPR also enables the resulting conformational change of the receptor to trigger a change in the bridged cantilever geometry.

[0205] A buffer in a biological system is to maintain intracellular and extracellular pH within a very narrow range and resist changes in pH in the presence of internal and external influences. The pH in a biological system controls the solubility, biological functions, and the chemical reactivity of biomaterials. The buffer maintains a pH that enables disassociation of analyte nucleic acid from its native complement or inherent secondary structure. Nonlimiting examples of the SPR buffer include Phosphate, MES, Bis-Tris Propane, 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, the SPR is between pH 7 - 11.

[0206] A detergent is a surfactant with an amphiphilic structure, where each molecule has a hydrophilic (polar) head and a long hydrophobic (non-polar) tail. The dual nature of the detergent facilitates the mixture of hydrophobic compounds with water, e.g., in a biological sample.

[0207] The SPR detergent can be an anionic detergent, a cationic detergent, a zwitterionic detergent, an anti-foaming detergent, or any combination thereof.

[0208] The low ionic strength detergent is capable of disrupting cell wall structures to release the analyte from a cell.

[0209] In some embodiments, the detergent comprises Triton X-100, TWEEN-20, NP-40, or Brij -surfactants. An oxidizing agent is a chemical species that tends to oxidize other substances, causing them to lose electrons and raises their oxidation state. The oxidizing agent in the SPR produces a controlled oxidation fragmentation of an analyte.

[0210] In some embodiments, the oxidizing agent includes oxygen, ozone, a peroxide compound, a nitrate compound, a nitrite compound, a chlorine compound, a perchlorate compound, a chlorate compound, a chlorite compound, a hypochlorite compound, a bromine compound, an iodine compound, a persulfate compound, a permanganate compound, a chromium compound, a chromate compound, a perborate compound, a bismuthate compound, a copper compound, an iron compound, a cerium compound, a lead compound, a silver compound, a rhenium compound, a hydroxylamine compound, a sulfur oxide compound, an osmium oxide compound, an N-oxide compound, a quinone compound, an amide compound, an imide compound, an cyanurate compound, a saccharin compound, a selenium compound, a perrhenate compound, a propyl gallate, or any combination thereof.

[0211] In specific embodiments, the oxidizing agent comprises a peroxide compound, a nitrate compound, a permanganate compound, or any combination thereof.

[0212] In some embodiments, the enzymatic activity inhibitor is present in the SPR. Nonlimiting examples of useful enzymatic activity inhibitors for the SPR include a chelating agent, an RNase inhibitor, a DNase inhibitor, a protease inhibitor, or any combination thereof.

[0213] A chelating agent is a chemical compound that reacts with metal ions to form stable, water-soluble metal complexes. The presence of the chelating agent in a biological sample protects nucleic acids from enzymatic degradation by removing the metal ions. The chelating agent also reduces interaction between proteins and nucleic acids, therefore enhancing the nucleic acids extraction efficiency in a biological sample. Nonlimiting examples of the SPR chelating agent comprises EDTA, EGTA, HEDTA, NTA, and TEA.

[0214] RNase inhibitors, DNase inhibitors, and protease inhibitors can also be present in the SPR to prevent analytes from being degraded in the process of using the SPR for detecting an analyte in a sample.

[0215] In additional embodiments, the SPR further comprises a chaotropic agent. A chaotropic agent disrupts the hydrogen bonding network in water solution, destabilizing the native state of macromolecules (e.g., proteins, nucleic acids) in the solution. The chaotropic agent denatures nucleic acids associated proteins, therefore weakening the hydrophobic interaction between nucleic acids associated proteins and nucleic acids. The chaotropic agent dissociates nucleic acids from nucleic acids associated proteins. Nonlimiting examples of the chaotropic agent comprises guanidinium thiocyanate, guanidine, urea, and thiourea.

[0216] A person having ordinary skill in the art would understand and be able to optimize the individual components in a sample processing reagent (SPR) for detecting an analyte in a sample.

[0217] Example 13: Detection of SARS CoV-2 by Bio-Cantilever Deflection

[0218] The 5’- and 3 ’-ends of Receptor DNA candidates were chemically modified to enable attachment to the gold surfaces of the cantilever sensors. Specifically, the 5’- phosphates were thiolated via standard alkaline phosphatase and T4 kinase reactions with ATPyS. The 3 ’-ends were modified via a T4-ligase reaction to a 3 ’-thiol modified oligonucleotide. Successful thiol modification was demonstrated by electrophoretic resolution of purified unmodified and thiol modified receptor DNA. In the absence of appropriate concentrations of reducing agent (i.e., DTT or TCEP), thiol modified Receptors form disulfide bonded multimers. Prior to bioassembly, the thiol-modified DNA ends were reduced with TCEP to eliminate thiol-thiol bonded multimers.

[0219] The cantilever body includes piezoresistive materials of which the conductivity or resistance changes as a function of cantilever deflection. The cantilever tip, and the support surface below the tip are gold coated to enable bio-attachment to the thiol-modified Receptors. Twenty silicon piezoresistive MEMS cantilevers were fabricated having various geometries and physical properties, e.g., materials, length, force constants, and Wheatstone bridge resistance values. Initially, two different lengths, 400 nm and 450 nm respectively, and two different tip designs were selected. The shorter cantilever possessed a sharp Si tip, while the longer version was tipless. FIG. 10 is a plot of the experimental details when testing various samples with the two cantilevers.

[0220] For initial testing the modules were mounted to a silicon wafer using double-sided Kapton tape. A second piece of double sided Kapton tape was used to shadow mask the sensor chip and mount. The plastic protectant was removed later only on the outside of a small (~1”) strip of tape. The wafer was placed on a substrate holder and was loaded into an electron beam evaporation system. The substrate rotation was turned off due to the distance between the masking tape to the cantilever tip that would have resulted in gold deposition under the tape. After depositing a Ti / Au stack (5 / 50 nm) at lA / s and 2A / s, respectively, the samples and the tape were removed. The BCSR bio-assembly and detection approach involved a series of steps to detect the presence of a complementary target molecule using cantilevers with attached receptors. This approach follows the general sequence shown in FIG. 9. First, the gold surfaces of the cantilever and the support base were prepared, followed by the creation of a gap between them. Voltage measurement was performed using a Data Acquisition (DAQ) system. Next, the gap was bridged by introducing a receptor, and an increase in the DAQ voltage was observed as the gap size increased, providing evidence of bridging. Unbound complementary strands were removed by flushing the system, and the formation of a singlestranded DNA bridge was demonstrated, which resulted in a change in the DAQ voltage. It was imperative to ensure that the voltage was distinct from the un-bridged gap voltage before bio-assembly. In the detection sequence, a sample that contained the complementary target molecule was introduced. The DAQ voltage was measured as an indicator of cantilever deflection due to the binding of the target molecule. Finally, these steps were repeated for additional detection testing.

[0221] To assess the sensitivity and specificity of the BCSR component assembly, nine distinct samples were evaluated with the DNA receptor 008. The nine samples are listed as follows: (1) Positive Control - complement: A positive control sample containing approximately 100 copies of 100% complementary DNA in sample preparation reagent (SPR) solution. (2) Negative Control - non-complement: A negative control sample containing approximately 100 copies of 40% complementary DNA in SPR solution. (3) COVID-19 RNA in SPR: Approximately 1000 copies of genomic RNA from SARS CoV- 2 strain 2019-nCoV / USA-WAl / 2020 (ATCC vr-1986d) was mixed with SPR solution. (4) COVID-19 virus in SPR: Approximately 1000 copies of heat-inactivated SARS-CoV-2 variant B. l.1.7 (ATCC vr-3326hk) was mixed with 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.l.1.7 (ATCC vr-3326hk) was mixed with SPR solution. (8) Non-COVID RNA in sputum-SPR: Approximately 1000 copies of genomic RNA from Human betacoronavirus OC43 (ATCC VR-1558D) in sputum-SPR solution. (9) Non-COVID virus in sputum-SPR: Approximately 1000 copies of Human betacoronavirus OC43 (ATCC VR-1558) in sputum- SPR solution. FIG. 10 is a bar graph showing the results of a BCSR bio-cantilever deflection in nanometers (nm) when detecting the nine samples listed above. The nine samples, with and without the COVID RNA, were tested in 10 replicates for their BCSR bio-cantilever deflection in nanometers (nm). The results showed that the positive samples containing the CO VID RNA produced a BCSR bio-cantilever deflection of around 30nm, while the negative samples lacking the COVID RNA exhibited a deflection of less than 2 nm, a significant difference from that of the positive samples. The results demonstrated the ability to detect the RNA as potential target analytes in the BCSR sensor. The results also demonstrated that removing the RNA from the RNA bound BCSR sensor was effective to “reset” the BCSR sensor. Legends in the graph: Comp = 100% complementary Target, Non-Comp = Target material with only 80% homology, CO VID RNA = purified RNA from Target virus, COVID virus = Target COVID SARS CoV-2 virus, Sputum = human sputum analog with no Target material, Non-COVID = non-COVID-19 coronavirus. Baseline, or physically disassociated BCSR cantilever deflection was consistently approximately Inm.

[0222] To further assess the reliability and the repeatability of the BCSR component assembly, three different cantilevers (black bar, grey bar, and hash bar) with the DNA receptor 008 were tested to detect four types of analytes (an exact nucleic acid receptor complement, a non-complementary nucleic acid, a genomic CO VID 19 RNA complement, and a non-COVID coronavirus genomic RNA.) The results, illustrated in FIG. 11, showed that all three BCSR sensors produced approximately identical cantilever deflections. Although each of the cantilevers had slightly different spring constants, the positive and negative detection results were consistent. The results demonstrated that the BCRS biocantilevers are reliable and can be consistently and robustly manufactured.

[0223] FIG. 12 is a table demonstrating the correlation of receptor inherent curvature to the magnitude of BCSR sensor response. Three DNA receptors (008, 012c, and 012s) were designed and tested against a complementary DNA for hybridization. The difference between 012c and 012s is a single nucleotide change. In the hybridization of the DNA receptor 008 and its complementary DNA, the calculated maximum reduction in end-to-end distance is 48 nm. Receptor 012c hybridization to its complement reduces the distance to 62 nm while receptor 012s hybridization to its complement reduces the distance to only 27 nm. Three-dimensional projections (XZ and XY perspectives) show the relative predicted curvature of each receptor-complement pair. In comparison, the measured cantilever deflection for receptor 008, receptor 012c, and 012s are 28nm, 32nm, and 17nm respectively. In each case, the magnitude of cantilever deflection is directly correlated to the calculated amount of inherent molecular curvature at 58 ± 5%. It is important to note that full hybridization between the complementary stands would be inhibited by the fact that the bridged receptor strand is permanently attached at both ends to the cantilever and stage respectively.

[0224] Example 14: Detection of SARS CoV-2 by Bio-Cantilever Deflection

[0225] Tests were conducted to evaluate the ability to detect multiple bioagent Targets including COVID, Influenza (H1N1), Adenovirus, and Human Rhinovirus (HRV). Specifically, the following receptors were targeted: COVID, H1N1 2c, H1N1 3a, Adeno_41, Adeno_49, Adeno_52, HRV A l 2, HRV B, and HRV C.

[0226] The table in FIG. 13 provides the details for the receptors that were designed and produced to detect Influenza A, Adenovirus B, and Human Rhinovirus (HRV). Two (2) to three (3) different candidate receptors were developed for each Target. This redundancy enables either selection of receptors with maximum detection performance, or the potential for multiplex systems where detection is based upon response to multiple markers of a target’s genome. Numerous bio-physical properties were considered as described in the table.

[0227] The columns of the table shown in FIG. 13 are defined as follows: “Source” of the receptors: the viral segment, specific NCBI / NIH database accession number, or consensus regions common to the most predominant variants of the target. (The corresponding data for each identified source can be readily found by a person of skill in the art on the world wide web at ncbi.nlm.nih.gov, which is incorporated herein by reference.) “Candidate”: internal nomenclature of a specific receptor candidate. “C&S”: the relative measure of curvature and stiffness of the receptor - larger values directly correlate to higher detection response. “ENDS”: a measure of the receptor curvature as calculated by the ratio of the actual length of the receptor along its helical axis compared to the end-to-end distance - larger values directly correlate to increased curvature. “% Reduction”: a more intuitive measure of receptor curvature as calculated by the actual molecule length compared to the end-to-end distance - larger values directly correlate to increased curvature. The % reduction can range widely, e.g., from about 15% up to nearly 80%. “AG”: Gibbs free energy is a measure of the potential for internal folding of the receptor molecule - larger values correlate to fewer issues with internal folding of target genome markers, thus inhibiting detection. Tm°C: a calculated measure of target genome fragment melting temperature that is critical with respect to appropriate denaturation and hybridization to the receptor. “Target Homology”: the calculated genetic match between the receptor and its target genome fragment - higher homology infers greater specificity. “Largest % homology to non-Target”: the percent match to the closest genetic variant, near-neighbor, or other microbe that may be found in the sample - Lower values infer greater specificity.

[0228] The identified Targets were selected to determine the effectiveness of the inventive approach to distinguish among multiple bioagents that induce cold and flu symptoms. The receptors were designed based upon the necessary biophysical properties including a high degree of specificity toward the desired detection Target and low homology to genetic nearneighbors. The bar chart results provided in FIGs. 14A-14I show the responses from five (5) piezo-resistive cantilever devices (solid or striped bars) each tested 10 times and reset between each test. The reset response consistently showed ~ 5nm + / - Inm as expected from experimental design on all tests. The vertical scales (nanometers) are all adjusted to lOOnm for chart-to-chart comparison. Note that deviations in individual Target biodetection response, i.e., cantilever deflection, are proportional to the calculated and independently validated Receptor inherent curvature. For example, the curvature induced reduction in end- to-end distance of the H1N1 2c Receptor (FIG. 14B) was predicted to be approximately 8 Inm. The measured cantilever deflection was approximately 50nm. On the other hand, cantilever deflection by the HRV-C Receptor (FIG. 141) was predicted to be 53nm, and the measured deflection was 33nm. In each case, the measured cantilever deflection was approximately 60-65 percent of the predicted reduction of Receptor length. This observation was not surprising due to the incomplete formation of the double-stranded helix, resulting in a lesser degree of structural change than predicted.

[0229] While the degree of deflection may not reach the predicted level, the deflection is nonetheless repeatably proportional to the predicted curvature and of sufficient magnitude for reliable detection. Each sample was tested at least ten times, with each of the Receptors, on multiple cantilevers. Test-to-test results are also very consistent. In summary, the binary result of IDC detection technology has been demonstrated to accurately and consistently detect viruses in the presence of a sputum simulant 10 out of 10 times within a matter of seconds. Example 15: Other embodiments

[0230] Item 1. A sensor assembly for detecting a target analyte, the sensor assembly comprising: a cantilever formed from a silicon material having a deflection detection element disposed therein, the cantilever being elongated and having a proximal end and a distal end; a support base having a silicon pedestal disposed on a support surface; a first conductive metallization disposed on a lower surface of the cantilever in electrical contact with the deflection detection element; a second conductive metallization disposed on an upper surface of the silicon pedestal, the second conductive metallization configured to conduct electrical signals between the deflection detection element and contacts in electrical communication with external electrical devices; a eutectic bond formed between the first and second conductive metallization and the pedestal, the eutectic bond configured to secure a proximal end of the cantilever on the pedestal, wherein the pedestal has a pedestal thickness to support a lower surface of the distal end of cantilever at a fixed gap from the support surface; and at least one bridge receptor having a first end and a second end, wherein the first end is configured for attachment to the lower surface of the distal end of the cantilever and the second end is configured for attachment to the support surface, wherein the bridge receptor is configured to change conformation upon interaction with the target analyte and induce deflection of the cantilever; wherein the deflection detection element generates an output signal indicative of deflection of the cantilever.

[0231] 2. The sensor assembly of item 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.

[0232] 3. The sensor assembly of item 2, wherein the plurality of piezoresistors are configured to define a Wheatstone bridge.

[0233] 4. The sensor assembly of item 1, wherein the eutectic bond comprises a goldsilicon bond.

[0234] 5. The sensor assembly of item 1, wherein the fixed gap is within a range of 1 - l,000nm. 6. The sensor assembly of item 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 the ends of the at least one bridge receptor.

[0235] 7. The sensor assembly of item 6, wherein the metallized contact area comprises gold and the at least one bridge receptor is thiol-modified to facilitate attachment to the metallized contact areas.

[0236] 8. The sensor assembly of item 1, wherein the at least one bridge receptor comprises an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

[0237] 9. The sensor assembly of item 8, wherein the at least one bridge receptor comprises a nucleic acid, a nucleic acid analogue, a double-stranded DNA (dsDNA), a dsDNA analog, a single-stranded DNA (ssDNA), a ssDNA analog, or a peptide nucleic acid.

[0238] 10. The sensor assembly of item 8, wherein the at least one bridge receptor is ssDNA.

[0239] 11. The sensor assembly of item 7, wherein the at least one bridge receptor is a peptide nucleic acid containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

[0240] 12. The sensor assembly of item 7, wherein the at least one bridge receptor is 50 - 1,000 nm in length.

[0241] 13. The sensor assembly of item 7, wherein the bridge receptor undergoes a reduction in length from about 15% to about 80% upon interaction with the target analyte.

[0242] 14. The sensor assembly of any one of items 1-13, 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 repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

[0243] 15. The sensor assembly of item 14, wherein the analyte comprises a complementary nucleic acid, a complementary nucleic acid analogue, a complementary dsDNA, a complementary dsDNA analog, a complementary ssDNA, a complementary ssDNA analog, a complementary RNA, or a complementary RNA analog.

[0244] 16. The sensor assembly of item 14, wherein the analyte is ssDNA.

[0245] 17. The sensor assembly of item 14, wherein the analyte is RNA.

[0246] Item 18. An analyte detection system comprising: a test well or chamber configured to receive the sensor assembly of any one of items 1-13; and conductive connectors configured for electrical communication between the sensor assembly and instrumentation for generating an external display indicative of detected deflection of the cantilever.

[0247] 19. The analyte detection system of item 18, wherein the test well or chamber is configured to retain a sample comprising one or more of a lachrymal fluid, saliva, a buccal fluid, a bronchoalveolar lavage fluid, mucus, a nasal sample, a nasopharyngeal sample, a breath sample, urine, feces, tissue, blood, plasma, serum, a cell culture, a bodily fluid, a tissue biopsy, an apocrine fluid, or an eccrine fluid, a forensic sample, an aerosol, soil, water sample, food, an ingredient, a raw material, an in-process sample, a byproduct, a product, or a quality control sample.

[0248] 20 . The analyte detection system of item 19, wherein the sample comprises a virus, a bacterium, a phage, a yeast, a mycoplasma, a fungus, a human cell, an animal cell, a plant cell, an insect cell, or any combination thereof.

[0249] 21. The analyte detection system of item 19, wherein the sample is a mucus sample, a nasal sample, a breath sample, or a nasopharyngeal sample.

[0250] 22. The analyte detection system of item 19, wherein the sample contains a virus.

[0251] 23. The analyte detection system of item 18, wherein the virus is one or more of a coronavirus, an influenza virus, a respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), or a zika virus.

[0252] The integrated NEMS cantilever sensing devices and approaches disclosed herein provide accurate, repeatable, and cost-effective tools for detecting pathogens and other biomarkers. The sensor unit can be fabricated at extremely low cost, 100 microns in size, and fractions of a milligram using established MEMS fabrication techniques and technology. Using the described exemplary fabrication processes, large-scale reliable, consistent reproducible, detection units with high specificity can be manufactured and employed for detection of analytes without the need for separation and purification processing. The optional resettable flow cell embodiment provides significant versatility with applications to clinical settings as well as large scale sample analysis as might be encountered in agriculture, food and water safety, and public health settings, e.g., schools and ports of entry. Unlike many existing diagnostic tests in which detection reagents have limited lifetimes, many elements of the inventive approach can be stockpiled (e.g., MEMS and electronic components) for extended periods without risk of degradation. The ability to rapidly sequence and synthesize DNA makes the inventive approach a realistic and cost- effective tool for preparedness for detection and containment in the event of future contagions.

Claims

What is claimed is:

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

5. The sensor assembly of claim 1, wherein the fixed gap is within a range of 1 - l,000nm.

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 the ends of the at least one bridge receptor.

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

8. The sensor assembly of claim 1, wherein the at least one bridge receptor comprises an inorganic molecule, an organic molecule, a polymer, a polymer analog, a carbohydrate, a carbohydrate analog, a nucleic acid, a nucleic acid analog, a protein, a protein analog, an antibody, an antibody analog, a repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

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

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

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

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

14. The sensor assembly of any one of claims 1-13, 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 repetition of any one of the molecules thereof, a conjugate of any one of the molecules thereof, a hybridization of any one of the molecules thereof, or any combination thereof.

15. The sensor assembly of claim 14, wherein the analyte comprises a complementary nucleic acid, a complementary nucleic acid analogue, 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 chamber configured to receive the sensor assembly of any one of claims 1-13; and conductive connectors configured for electrical communication between the sensor assembly and instrumentation for generating an external display indicative of detected deflection of the cantilever.

19. The analyte detection system of claim 18, wherein the test well or chamber is configured to retain a sample comprising one or more of a lachrymal fluid, saliva, a buccal fluid, a bronchoalveolar lavage fluid, mucus, a nasal sample, a nasopharyngeal sample, a breath sample, urine, feces, tissue, blood, plasma, serum, a cell culture, a bodily fluid, a tissue biopsy, an apocrine fluid, or an eccrine fluid, a forensic sample, an aerosol, soil, water sample, 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 phage, 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), adenovirus, human rhinovirus (HRV), or a zika virus.

Images