Integrated Silicon Platform for Electronic Biosensors

Abstract

A structure, an apparatus, and a method are disclosed. A silicon substrate including a microfluidic system that receives a sample fluid and prepares an analyte solution received by a reservoir including a sensing surface is electrically connected to a semiconductor device. The structure includes components for receiving the sample fluid, components for preparing the sample fluid, a CRISPR system for splitting a reporter species when the sample fluid contains a sequence of interest, a sorting component for separating the split reporter species from the sample fluid and the CRISPR product, a cavity for receiving the analyte solution containing the split reporter species, and a sensing surface within the cavity. The sensing surface detects an electrical signal induced by a reaction event in the analyte solution.

Description

[Technical field]

[0001] The present disclosure relates to electronic biosensors, and more particularly, to CRISPR-based electronic sensors integrated with semiconductor devices. [Background technology]

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems act as molecular scissors for RNA and DNA molecules. CRISPR-based diagnostic systems rely primarily on the identification of nucleic acid sequences of interest associated with a given disease, which trigger secondary cleavage events of non-specific reporter molecules to produce a detectable result. For example, the reporter molecules can generate fluorescent or colorimetric signals. Summary of the Invention

[0003] Various embodiments are directed to a structure including a component for receiving a sample fluid, a component for preparing the sample fluid, a CRISPR system for splitting a reporter species when the sample fluid includes a sequence of interest, and a sorting component for separating the split reporter species from the sample fluid and the CRISPR product. In some embodiments, the reporter species includes a nucleic acid, a reporter ligand attached to a first end of the nucleic acid, and a bead attached to a second end of the nucleic acid. The sorting component can include a filter having a pore diameter smaller than the bead. In some embodiments, the bead can be magnetic. In these embodiments, the sorting component can include a magnet. In some embodiments, the sorting component includes a deterministic lateral displacement array. The structure further includes a cavity for receiving an analyte solution including the split reporter species, and a sensing surface within the cavity. The sensing surface detects an electrical signal induced by a reaction event in the analyte solution. The reaction event can include chemical bond formation between the reporter species and a molecule coating the sensing surface. The structure further includes a semiconductor device, such as a field effect transistor (FET) device or a bipolar junction transistor (BJT) device, electrically connected to the sensing surface. The structure may further include a microfluidic channel.

[0004] Further embodiments are directed to an apparatus including a semiconductor device and a silicon substrate having one or more microfluidic systems. The semiconductor device can be, for example, a FET device, a BJT device, or a nanowire device. In some embodiments, the silicon substrate has multiple microfluidic systems in individual wells. Each microfluidic system includes an inlet through which a sample fluid can flow, a cavity containing a reporter species, and a CRISPR system that splits the reporter species when a sequence of interest is present in the sample fluid. Each microfluidic system further includes components for separating the split reporter species into an analyte solution, a sensing surface electrically connected to the semiconductor device, and a reservoir in which the analyte solution contacts the sensing surface. The reservoir includes components for inducing a reaction event when the split reporter species is present. In some embodiments, each microfluidic system has a fluid gate controlled by an embedded electrode.

[0005] Further embodiments are directed to a method including providing one or more microfluidic systems on a silicon substrate. In some embodiments, the silicon substrate has multiple microfluidic systems in individual wells. Each microfluidic system includes a component for receiving a sample fluid, a component for preparing the sample fluid, a CRISPR system for splitting a reporter species when the sample fluid includes a sequence of interest, and a sorting component for separating the split reporter species from the sample fluid and the CRISPR product. In some embodiments, the reporter species includes a nucleic acid, a reporter ligand attached to a first end of the nucleic acid, and a bead attached to a second end of the nucleic acid. The CRISPR ligand can be preloaded in the sample preparation cavity. The structure further includes a cavity for receiving an analyte solution including the split reporter species, and a sensing surface within the cavity. The cavity can include a reservoir in which the analyte solution contacts the sensing surface. The reservoir can include a component for inducing a reaction event upon the presence of the split reporter species. The sensing surface detects an electrical signal induced by a reaction event in the analyte solution. Each microfluidic system may include a fluidic gate controlled by the embedded electrode. The method further includes providing a semiconductor device, such as a field effect transistor (FET) device or a bipolar junction transistor (BJT) device, electrically connected to the sensing surface.

[0006] The drawings contained in this application are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The drawings are merely illustrative of certain embodiments and are not intended to limit the present disclosure. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a block diagram illustrating a biosensor device according to some embodiments of the present disclosure. [Diagram 2]FIG. 1 is a schematic diagram illustrating a cross-sectional view of a microfluidic biosensor structure according to some embodiments of the present disclosure. [Diagram 3] FIG. 1 is a schematic diagram illustrating the process of secondary resolution of reporter species according to some embodiments of the present disclosure. [Figure 4A] 1 is a schematic diagram illustrating a cross-sectional view of an exemplary separation channel, according to some embodiments of the present disclosure. [Figure 4B] 1 is a schematic diagram illustrating a cross-sectional view of an exemplary separation channel, according to some embodiments of the present disclosure. [Figure 4C] 1 is a schematic diagram illustrating a cross-sectional view of an exemplary separation channel, according to some embodiments of the present disclosure. [Diagram 5] 1 is a schematic diagram illustrating an example detection module structure, according to some embodiments of the present disclosure. [Figure 6] 1 is a schematic diagram illustrating an example detection module structure, according to some embodiments of the present disclosure. [Figure 7] 1 is a schematic diagram illustrating a biosensor structure including multiple microfluidic systems on a substrate according to some embodiments of the present disclosure. [Figure 8A] 1 is a schematic diagram illustrating a structure including a well of a battery according to some embodiments of the present disclosure. [Figure 8B] 1 is a schematic diagram illustrating a structure including a well of a battery according to some embodiments of the present disclosure. [Figure 8C] 1 is a schematic diagram illustrating a structure including a well of a battery according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] While the invention is subject to various modifications and alternative forms, details thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that it is not intended to limit the invention to the particular embodiments described. Instead, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

[0009] Embodiments of the present invention are generally directed to The present disclosure is not necessarily limited to such applications, and various aspects of the disclosure will be understood by discussing various examples with this context.

[0010] Various embodiments of the present disclosure are described herein with reference to the associated drawings. Alternative embodiments may be devised without departing from the scope of the present disclosure. It should be noted that various connections and relationships (e.g., above, below, adjacent, etc.) are specified between elements in the following description and drawings. These connections and / or relationships may be direct or indirect unless otherwise specified, and the present disclosure is not intended to be limiting in this respect. Thus, the coupling of entities may refer to either direct or indirect coupling, and the relationship between entities may be a direct or indirect relationship. As an example of an indirect relationship, in this description, reference to forming layer "A" on layer "B" includes the situation where one or more intermediate layers (e.g., layer "C") are between layer "A" and layer "B", as long as the relevant features and functionality of layer "A" and layer "B" are not substantially altered by the intermediate layer(s).

[0011] The following definitions and abbreviations will be used for interpretation of the claims and the specification. As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "containing," or "containing" or any other variations thereof are intended to cover a non-exhaustive inclusion. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to those elements, but can include other elements that are not expressly listed or that are inherent to such composition, mixture, process, method, article, or device. Additionally, the word "providing" as used herein can refer to various actions such as making, purchasing, obtaining, synthesizing, making available, and the like, or combinations thereof.

[0012] For the purposes of the following description, the terms "above," "below," "right," "left," "vertical," "horizontal," "top," "bottom," and derivatives thereof, refer to the described structures and methods oriented in the depicted drawings. The terms "above," "atop," "on top of," "on top of," "disposed on," or "disposed on top of" mean that a first element, such as a first structure, is on a second element, such as a second structure, and an intervening element, such as an interface structure, may be between the first and second elements. The term "direct contact" means that a first element, such as a first structure, and a second element, such as a second structure, are connected without any intermediate conductive insulating or semiconducting layer at the interface between the two elements. It should be noted that the term "selective to," such as, for example, a "first element selective to a second element," means that the first element can be etched and the second element can act as an etch stop.

[0013] As used herein, the articles "a" and "an" preceding an element or component are intended to be open-ended with respect to the number of instances (i.e., occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and singular word forms of the element or component also include plurals, unless the number is clearly meant to be singular.

[0014] As used herein, the term "invention" or "the present invention" is an open-ended term and is not intended to refer to any single embodiment of a particular invention, but to encompass all possible embodiments and / or combinations of embodiments described in the specification and claims.

[0015] Unless otherwise stated, ranges (e.g., time, concentration, temperature, etc.) set forth herein include both the endpoints and all numbers between the endpoints. Unless otherwise specified, the use of terms such as chilled (~), or "about," "approximately," "approximately," "slightly less than," and variations thereof, is intended to include the degree of error associated with the measurement of a particular quantity based on the equipment available at the time of filing. For example, "about" can include a range of ±8% or 5% or 2% of a given value, range of values, or one or more endpoints of a range of values. Unless otherwise indicated, the use of such terms in conjunction with ranges applies to both ends of the range (e.g., "approximately 1g to 5g" should be interpreted as "approximately 1g to approximately 5g") and in conjunction with a list of ranges applies to each range in the list (e.g., "approximately 1g to 5g, 5g to 10g, etc." should be interpreted as "approximately 1g to about 5g, about 5g to about 10g, etc.").

[0016] It should be further understood that material compounds will be described in terms of the listed elements, e.g., SiN or SiGe. These compounds may contain different percentages of the elements within the compound, e.g., SiGe may contain Si x Ge (1-x)where x is less than or equal to 1, etc. Additionally, other elements can be included in the compound and still function according to the present principles. Compounds that include additional elements will be referred to herein as alloys.

[0017] For the sake of brevity, conventional techniques for semiconductor device and integrated circuit (IC) manufacturing may or may not be detailed herein. Additionally, various tasks and process steps described herein may be incorporated into more comprehensive procedures or processes having additional steps or functions not detailed herein. In particular, the various steps of the fabrication of semiconductor devices and semiconductor-based ICs are well known, and therefore, for the sake of brevity, many conventional steps will be mentioned only briefly herein or will be omitted entirely without detailing the well-known processes. It should be noted that not all of the masking, patterning, and lithography processes are shown, as one of ordinary skill in the art would understand where masking and patterning processes are utilized to form the specified layers and openings and perform the specified selective etching processes as described herein.

[0018] Generally, the various processes used to form the microchips that are packaged to become ICs fall into four general categories: thin film deposition, removal / etching, semiconductor doping, and patterning / lithography.

[0019] Deposition is any process whereby material is grown, capped, or otherwise transferred onto a wafer. Available techniques include physical vapor deposition (PVD), chemical vapor deposition (CVD), electrochemical deposition (ECD), molecular beam epitaxy (MBE), and more recently atomic layer deposition (ALD), among others. Another deposition technique is plasma-enhanced chemical vapor deposition (PECVD), a process that uses energy in a plasma to induce reactions at the wafer surface that would otherwise require higher temperatures associated with conventional CVD. The bombardment of energetic ions during PECVD deposition can further improve the electrical and mechanical properties of thin films.

[0020] Removal / etching is any process that removes material from a wafer. Examples include etching processes (either wet or dry) and chemical mechanical planarization (CMP). One example of a removal process is ion beam etching (IBE). In general, IBE (or milling) refers to dry plasma etching methods that utilize a remote broad beam ion / plasma source to remove substrate material by physically inert gas or chemically reactive gas means or both. Like other dry plasma etching techniques, IBE offers advantages such as etch rate, anisotropy, selectivity, uniformity, aspect ratio, and minimal substrate damage. Another example of a dry removal process is reactive ion etching (RIE). In general, RIE uses a chemically reactive plasma to remove material deposited on the wafer. With RIE, a plasma is generated by an electromagnetic field under low pressure (vacuum). High energy ions from the RIE plasma attack the wafer surface and react with it to remove the material.

[0021] Semiconductor doping is the modification of electrical properties by doping, for example, the source and drain of a transistor, typically by diffusion or by ion implantation or both. These doping processes are followed by furnace annealing or rapid thermal annealing ("RTA"). The annealing serves to activate the implanted dopants. Thin films of both conductors (e.g., polysilicon, aluminum, copper, etc.) and insulators (e.g., various forms of silicon dioxide, silicon nitride, etc.) are used to connect and insulate transistors and their components. Selective doping of different regions of a semiconductor substrate can change the conductivity of the substrate with the application of a voltage. By creating structures of these various components, millions of transistors can be constructed and wired together to form the complex circuits of modern microelectronic devices.

[0022] Semiconductor lithography is the formation of a three-dimensional relief image or pattern on a semiconductor substrate for subsequent transfer of the pattern to the substrate. In semiconductor lithography, the pattern is formed by a light-sensitive polymer called photoresist. The lithography and etching pattern transfer steps are repeated multiple times to build the complex structures that make up the transistors and the many wires that connect the millions of transistors in a circuit. Each pattern printed on the wafer is aligned with previously formed patterns, gradually building up conductors, insulators, and selectively doped regions to form the final device.

[0023] Proceeding now to an overview of the technology more specifically related to the embodiments of the present disclosure, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system acts as molecular scissors for RNA and DNA molecules. CRISPR-based diagnostic systems rely primarily on the identification of disease-associated sequences of interest that trigger secondary cleavage events in non-specific reporter molecules to produce fluorescent or colorimetric signals that can be read. These assays have been used to detect viruses such as Dengue, Zika, HPV, and COVID-19. Although CRISPR-based diagnostic systems can achieve attomolar sensitivity with very high specificity, the assay run time of the system is hampered by the need for very long sample preparation steps and trained laboratory professionals to produce the sequences of interest. For example, clinical samples such as urine, saliva, and blood require extraction of the nucleic acid of interest by processing steps such as centrifugation, sample disruption, etc.

[0024] The embodiments of the present disclosure can overcome these and other problems. The embodiments include a sample-to-answer CRISPR-based biosensor that can detect sequences of interest present in a sample fluid using a microfluidic system integrated with semiconducting devices (e.g., bipolar junction transistors (BJTs), field effect transistors (FETs), or nanowires). When the sample fluid contains a sequence of interest, the CRISPR system creates a reporter molecule that can be electronically detected based on an electrical signal caused by, for example, a change in pH or chemical bond formation. Electrical sensors can be more sensitive than optical detectors. This can allow for the use of smaller sample volumes without the need to extract and amplify the nucleic acid of interest.

[0025] Additionally, the disclosed sensors offer compatibility with silicon technology for scaling, miniaturization, and automation. For example, modules for pre-detection sample processing, enrichment, and filtering of samples can be integrated with silicon technology. Silicon fabrication further allows for miniaturization and high-throughput sensing in a smaller footprint compared to optical sensors. Compartments (e.g., reservoirs, channels, etc.) of the disclosed microfluidic biosensors can be optimized to allow for the use of low sample volumes. For example, MEMS-based microfabrication techniques can be used to miniaturize and scale the platform. In further embodiments, the disclosed biosensors can be integrated at the macroscale by spot-drying reagents onto paper and using wax printing to create microfluidic channels that lead to a sensing surface that is electrically connected to a semiconductor device.

[0026] It should be understood that the foregoing advantages are exemplary advantages and should not be construed as limiting, and that embodiments of the present disclosure may include all, some, or none of the foregoing advantages while remaining within the spirit and scope of the present disclosure.

[0027] Referring now to the drawings, in which like numbers represent the same or similar elements, FIG. 1 is a block diagram illustrating components of a biosensor 100, according to some embodiments of the present disclosure. The biosensor 100 can include a microscale and / or macroscale fluidic system integrated with semiconductor technology. The biosensor includes a sample processing module 110, a separation module 120, and a detection module 130. The sample processing module 110 can include sample preparation reagents, a CRISPR system, and a reporter species. The separation module 120 can include components for separating secondary division products to create an analyte fluid that may include a divided reporter species. The detection module 130 can include a reservoir for the analyte fluid, having a sensing surface electrically connected to a semiconductor device. The modules 110, 120, and 130 can include various structures (e.g., channels, filters, reservoirs, electrodes, via holes, etc.) and reactants (e.g., CRISPR-associated enzymes, reporter species, and other reagents), etc., which are discussed in much more detail with respect to FIGS. 2-8(C).

[0028] The platform may be integrated with semiconductor technology on a macroscale by, for example, spot drying reagents onto paper that may be part of the sample processing module 110 and using wax printing to create microfluidic channels. The biosensor 100 platform may also be integrated with semiconductor technology on a microscale (e.g., by employing MEMS-based microfabrication techniques to miniaturize and scale the platform).

[0029] FIG. 2 is a schematic diagram illustrating a cross-sectional view of a microfluidic biosensor structure 200 according to some embodiments of the present disclosure. The structure 200 can be a microfluidic device including modules 110, 120, and 130 of FIG. 1. The components of these modules 110, 120, 130 (e.g., channels, reservoirs, electrodes, conductive contacts to semiconductor devices, etc.) can be patterned and embedded in a substrate 203, such as a silicon wafer. However, in other embodiments, the substrate 203 can be made of another material, such as quartz. The material of the substrate 203 can be any suitable material or combination of materials known to those skilled in the art that supports lab-on-a-chip or other microfluidic device fabrication processes, which can be a single layer or multiple sublayers.

[0030] The illustrated structure 200 may have a cover 206 layer that provides protection from the external environment. In some embodiments, at least a portion of the cover layer 206 is made of a transparent material, such as glass or polymethylmethacrylate. A transparent cover layer 206 may allow for more complex pass criteria or evaluations and avoid falsely-contrary results (e.g., by redirecting the analyte droplet to the sensing surface or by ensuring proper alignment and residence time of the analyte droplet over the sensing surface).

[0031] There may be via holes 210A, 210B, 210C, and 210D (collectively 210) through the substrate 203. The vias 210 may be defined by RIE or related processes that allow for loading of samples (e.g., via via 210A) and reagents (e.g., via vias 210B and 210C) as well as connection of electrical components (e.g., via via 210D). Loading of reagents may include printing, injecting, vapor deposition, mechanical insertion, taping, or dropping of reagents into the vias 210B and 210C. The reagent loading into the vias 210B and 210C may then be sealed from the backside by a substantially impermeable and mechanically robust thin film or membrane 226 to prevent loss or damage (e.g., leakage, oxidation, etc.) of the reagents. As used herein, "backside" refers to the side of the substrate 203 opposite the cover layer 206. Sealing can include chemical bonding to silicon / silica at the substrate 203 surface, mechanical bonding by compression or clamping, heat sealing, etc. Reagents can be loaded in any phase (gas, liquid, solid, etc.) and can be prevented from mixing or migrating into adjacent cavities when fluid gates 216 (see below) are formed and closed prior to loading. In some embodiments, via holes 210 can be temporarily and selectively unmasked during reagent loading to allow different stage sets to be filled with different reagent packages.

[0032] Additional layers / structures (not shown) may be included in the biosensor 200. For example, in some embodiments, there may be an interconnect layer for a multi-channel device. In another example, the biosensor structure 100 / 200 may include a three-dimensional fluidic network for distribution of samples and reagents. There may be multiple reactor wells on the chip / substrate 203 to perform simultaneous testing processes. For example, there may be an array of biosensors 100 in individual wells (see, for example, Figures 8(A)-8(C)). Various samples or other fluids / solutions may be introduced into the wells via channels formed on the same layer as the wells or from an upper layer formed in an additional lithography step.

[0033] The sample processing module 110 can include a sample loading inlet 213. The inlet 213 can include a cavity (channel or reservoir or both) for receiving a sample. The sample can be loaded through a via hole 210A that leads to the external environment and is drawn from the via hole 210A. In some embodiments, the sample is loaded using a pipette, syringe, or the like. In further embodiments, the sample fluid can be introduced by immersing all or a portion of the biosensor 200 in the fluid. For example, the biosensor 200 can be mounted (e.g., embedded, glued, suspended, etc.) within an environment such as a pond or other body of water, soil, a living organism, etc. The biosensor 200 can further be incorporated into the packaging or shell of a machine or instrument for interfacing with a surface, encapsulated in a pill or intravenous mechanism, etc. In some embodiments, the biosensor 200 can be mounted on a probe station or stylus used to position the device (e.g., robotically).

[0034] The sample fluid may be trapped in the inlet 213 cavity by a fluid gate 216A (see below) before flowing further into the sample processing module 110. In some embodiments, the sample processing module 110 includes a size filter 215 disposed between the inlet 213 and the first fluid gate 216A to remove cellular debris, large proteins, etc. from the sample fluid. Movement of fluid through the device may be facilitated by passive transport using various microfluidic techniques known in the art (e.g., capillary pumps). Active transport techniques may also be used in some embodiments. While specific capillary pump channels / structures are not shown herein, it will be understood by one of skill in the art that various channel configurations may be formed using the patterning techniques (e.g., RIE) discussed in more detail above.

[0035] Each stage of the sample testing process may be isolated from the upstream / next-stage process by liquid-actuated valves / gates ("fluid gates") 216A, 216B, 216C, and 216D (collectively 216), which act as a barrier between structures (e.g., channels and reservoirs) when closed. In some embodiments, the biosensor 200 may include a greater or lesser number of fluid gates 216. The timing of opening of each fluid gate 216 may be preset to stop the flow of the liquid phase until the completion of the action at a given stage. Various techniques may be used to open the fluid gates 216 at the appropriate time. For example, the fluid gates 216 may have a barrier that can be dissolved by the sample fluid. This may be particularly beneficial for single-use biosensors, where fluid gates 216 that can be repeatedly opened and closed and may be more expensive are unnecessary.

[0036] In some embodiments, at least one of the fluid gates 216 is electronically controlled. This may allow the sample testing process to be digitally defined, activated, and optionally guided by feedback (e.g., from a sensor(s) and / or user input commands). The electrical signal may be a direct current (DC) voltage controlled by an external source and / or on-chip electronics. In some embodiments, the fluid gates 216 may be actuated by applying Joule heating to melt or otherwise rupture a barrier material. The electrical signal may further effect barrier removal by piezoelectricity, electrostriction, etc. In some embodiments, at least one of the fluid gates 216 may be a capillary pinning location that may be reversibly actuated by applying an electrical signal to change the surface tension of the fluid at the pinning location.

[0037] Electrodes (not shown) for actuating the fluid gate 216 may be embedded in the substrate 203 using conventional semiconductor processing techniques (e.g., damascene processes). In some embodiments, the electrodes are copper (Cu). However, any material capable of serving as an electrode (e.g., aluminum (Al), titanium (Ti), tantalum (Ta), titanium nitride (TiN), tantalum nitride (TaN), tungsten (W), etc.) may be used. In some embodiments, at least a portion of each electrode may be overcoated with a more inert material such as a noble metal (e.g., gold (Au), platinum (Pt), rhodium (Rh), palladium (Pd), etc.). This may improve compatibility of the electrodes with biological samples and fluids by providing protection from damage due to oxidation / corrosion, erosion, impurities, etc.

[0038] The sample processing module 110 may further include a sample preparation cavity 217. When the fluid gate 216A is opened, the sample fluid may flow into the sample preparation cavity 217, which may include at least one channel. The sample preparation cavity 217 may further include one or more reservoirs. Reactants for the sample processing step are present in the sample preparation cavity 217. These reactants may include sample preparation reagents, reporter species (see FIG. 3), and a CRISPR-Cas system ("CRISPR system"), and may be preloaded into the cavity 217 through the via hole 210B. The preloaded reactants may be substantially dry before the sample flows in. For example, the reactants may be at least dry / sufficiently concentrated to delay or prevent the reactants from reacting, decomposing, etc., before being dissolved in the sample fluid.

[0039] The sample preparation reagents may include disruptive reagents (e.g., for disrupting cellular components) and stabilizing reagents to prevent degradation of nucleic acids. The sample preparation cavity 217 may include one further additional preparation component not shown here, such as an electrokinetic mixer, a component for increasing the concentration of the sample, a heating device, etc. These preparation components may allow the sample to be directly loaded into the biosensor 200 without the need for additional equipment or preparation steps. However, one or more preparation steps (e.g., mixing, lysis, etc.) may also be performed prior to loading of the sample in some embodiments.

[0040] The CRISPR system in the sample preparation cavity 217 contains a guide RNA or DNA ("RNA / DNA") that is complementary to a sequence of interest. Binding of the sequence of interest to the guide RNA / DNA activates a CRISPR-associated endonuclease protein (e.g., Cas9, Cas12, Cas13, etc.). This activation triggers a secondary nuclease activity ("secondary cleavage") in which the non-specific reporter RNA / DNA is cleaved at a known specific amino acid sequence. The CRISPR system may be consistent with any suitable CRISPR technology known to those of skill in the art (e.g., SHERLOCK (Specific High Sensitivity Enzyme Reporter UnLOCKing), DETECTR (DNA Endonuclease Targeted CRISPR Trans-Reporter), etc.). When the sequence of interest is present in the sample, the resulting secondary cleavage may create a cleaved reporter molecule for subsequent detection. This is illustrated in FIG. 3.

[0041] FIG. 3 is a schematic diagram illustrating a process 300 of secondary splitting of a reporter species 303 according to some embodiments of the present disclosure. The reporter species 303 includes a bead 313 (e.g., a micro- or nanoparticle) bound to a first end of a reporter nucleic acid ("reporter sequence") 316 and a reporter ligand 319 bound to a second end of the reporter sequence 316. Examples of reporter ligands 319 can be biomolecules such as biotin, DNA fragments, enzymes, etc. In some embodiments, the reporter ligand 319 is an enzyme embedded in DNA origami or surface-bound to a nanoparticle. The process 300 results in split beads 306 and split reporter molecules ("split reporters") 309. The split beads 306 include beads 313 bound to segments 317 of split reporter nucleic acid 316. The split reporter 309 comprises the remaining segment 318 of the split nucleic acid 316 bound to a reporter ligand 319. The split reporter 309 can be separated from the split beads 306 and the remaining unsplit reporter species 303 in the separation module 120.

[0042] The separation module 120 of the structure 200 shown in Figure 2 includes a separation channel 218 that may be separated from the sample processing cavity 217 by a fluid gate 216B. The separation module 120 may further include additional channels and / or reservoirs, at least one sorting component for separating beads (e.g., split beads 306 and non-split reporters 303) from split reporters 309, and an exit via. Examples of separation module 120 components that may be used in the separation channel 218 are discussed in more detail with respect to Figures 4(A)-(C).

[0043] 4(A), 4(B), and 4(C) show cross-sectional views 400, 410, and 420 of example separation channels 403, 413, and 423, according to some embodiments of the present disclosure. Separation channels 403, 413, and 423 are examples of separation channels that may be included in separation module 120 (FIG. 1). In some embodiments, separation channel 218 of biosensor 200 (FIG. 2) may be substantially similar to separation channels 403, 413, or 423. However, any suitable separation structure / technique may be used to separate the split reporter from the split beads and non-split reporter. In FIGS. 4(A)-(C), mixtures 409, 419, and 429 include species that are substantially the same as or similar to reporter species 303, split beads 306, and split reporters 309 shown in FIG. 3. The flow direction of the species in mixtures 409, 419, and 429 is indicated by the bold arrows.

[0044] 4(A) shows a separation channel 403 that includes a magnetic sorting component 406. The magnetic sorting component 406 can be used to separate species in a mixture 409 by magnetic beads. For example, the unsplit reporter species 303 and the split beads 306 can have magnetic beads 313. The magnetic sorting component 406 can attract and hold the magnetic unsplit reporter species 303 and the split beads 306 while allowing the split reporter species 309 to pass through the channel 403.

[0045] FIG. 4(B) shows a separation channel 413 that includes a size selection component 416. The size selection component 416 can be a size filter with pores smaller than the diameter of the beads 313 of the species 303 and 306 in the mixture 419. The size filter can be used to capture magnetic or non-magnetic beads. The split beads 306 and the unsplit reporter 303 are detained by the size filter, while the split reporter 309 can pass through the separation channel 413.

[0046] FIG. 4(C) shows a separation channel 423 that sorts based on size using a deterministic lateral displacement (DLD) array. The separation channel 423 can include a pillar array sorting component 426. For example, the sorting structure 426 can be a fluidic colloidal separation structure based on a nano- or micro-pillar array. The pillar array of the sorting structure 426 is represented in FIG. 4(C) by a gray and white diamond pattern. This is for illustrative purposes and is not intended to precisely depict the colloidal separation structure used for DLD sorting, as will be understood by those skilled in the art. In the sorting channel 423, a pillar array 426 can divert particles over a maximum size (unsplit reporter species 303 and split beads 306) to a waste channel, outlet, or reservoir 427, or a combination thereof, while smaller species (split reporters 309) pass through the sorting channel 426 into a next cavity 428, which may be a component of the detection module 130. In some embodiments, the pillar array 426 can include pillars having sensing surfaces electrically connected to the semiconductor device 209.

[0047] It should be understood that the separation channels shown in Figures 4(A)-(C) are illustrative examples, and any suitable separation mechanism known to one of skill in the art that is not otherwise inconsistent with this disclosure is contemplated. Furthermore, any number of separation channels and any combination of separation channels can be used. For example, a separation module can include both a magnetic separation channel 403 and a size-selection separation channel 413.

[0048] Returning again to FIG. 2, the fluid portion passing through separation channel 218 (e.g., separation channels 403, 413, or 423 or a combination thereof) is referred to herein as the "analyte solution" and is tested for the presence of split reporter 309 in detection module 130. In FIG. 2, detection module 130 of biosensor 200 includes detector cavity 219. The analyte solution travels through fluid gate 216C and enters detector cavity 219. Detector cavity 219 can optionally include an electrokinetic component (not shown) to increase the concentration of split reporter molecules, which can improve the signal-to-noise ratio during electrical signal monitoring.

[0049] The detector cavity 219 can further comprise reactants for inducing a reaction event that can be electronically detected when the split reporter is present in the analyte solution. In some embodiments, the reagents can be preloaded through backside via holes 210C (see above). The reagents in the detector cavity 219 can be any material suitable for the type of reporter ligand for the split reporter.

[0050] The detector cavity 219 can be temporarily separated from the sensing reservoir 223 by the fluid gate 216D in some embodiments. However, in other embodiments, the detector cavity 219 and the sensing reservoir 223 can be combined, for example, into a single reservoir. The timing of the fluid gate 216D can further be adjusted based on the type of reaction event to be detected at the sensing surface 233. For example, when the reporter ligand is a DNA fragment, the fluid gate 216D can be opened after a period during which an isothermal amplification reaction is performed in the detector cavity 219. Furthermore, the split reporter, including the biotin reporter ligand, can proceed directly from the separation channel 218 to the streptavidin-modified sensing surface 233. In another example of a detectable binding event in which the reporter ligand is a DNA fragment, the sensing surface 233 can be coated with a complementary DNA strand.

[0051] In further embodiments, the sensing surface 233 can detect the presence of the reporter ligand based on a difference in current caused by a pH change and / or a charge change. In these instances, the sensor material can be a material for reacting with the ligand to induce a pH change and / or a charge change. For example, if the reporter ligand includes an enzyme (e.g., glucose oxidase or horseradish peroxidase), the sensor cavity 219 or the sensor reservoir 223 or both can include an appropriate enzyme substrate (e.g., glucose). Binding of the substrate can result in an enzyme-catalyzed reaction that changes the pH and charge of the solution. When the reporter ligand includes DNA origami, a pH change and / or a charge change of the analyte solution can be further caused by the sugar phosphate backbone of the DNA. In another example, if the reporter ligand is a DNA fragment, the split reporter can be mixed with an isothermal amplification reagent. This can induce a pH change and / or a charge change at room temperature by multiplying the DNA fragment.

[0052] Chemical bond formation and / or pH change on / near sensing surface 233 can be detected by semiconductor device 209 when a voltage is applied to the analyte solution in sensor reservoir 223 by a reference electrode (see FIGS. 5 and 6). Sensing surface 233 can have any suitable structure for detecting electrical signals in the solution. In some embodiments, surface 233 can be wires, two- or three-dimensional rectangular or rounded structures, an array of pillars, etc. Sensing surface 233 can be electrically connected to semiconductor device 209 by conductive metal traces 236 and connectors 239 through backside wiring vias 210D. Examples of metals that may be used to form the conductive metal traces 236 include silver (Ag), gold (Au), tungsten (W), nickel (Ni), titanium (Ti), molybdenum (Mo), tantalum (Ta), copper (Cu), platinum (Pt), ruthenium (Ru), iridium (Jr), rhodium (Rh), and rhenium (Re), and alloys thereof. However, any suitable conductive material (e.g., metal nitrides, silicides, etc.) may be used. The connectors 239 may be conductive metal solder balls in some embodiments. However, any suitable connectors 239, such as metal studs, may be used. The semiconductor devices 209 connected to the sensing surface 233 may be field effect transistors (FETs) or bipolar junction transistor (BJT) devices (see, e.g., FIGS. 5 and 6). In some embodiments, the FET devices are silicon nanowire FETs. However, any FET device suitable for microfluidic applications may be used.

[0053] In some embodiments, the detection module 130 can be coupled with optical sensing modalities (e.g., colorimetry, fluorescence, etc.) to provide further data indicative of the presence or absence of the sequence of interest. These examples may include reporter species compatible with optical sensing. These may include any suitable reporter for CRISPR, including optical sensing. In some embodiments, the same reporter ligand may be detected optically as well as electrically. The optical modality may be part of an integrated on-chip optoelectronic system. The optoelectronic system may include components such as sensors, emitters, photonics, waveguides, filters, splitters, and modulators for translating optical signals into electronic signals.

[0054] 5 and 6 are schematic diagrams illustrating example detection module structures 500 and 600 according to some embodiments of the present disclosure. Structures 500 and 600 may be components of detection module 130 and may include semiconductor devices 503 and 603 (BJT devices 503 and FET devices 603), sensing surfaces 506 and 606, and sensor reservoirs 509 and 609. Sensing surfaces 506 and 606 may be optionally functionalized with biomolecules. In some embodiments, semiconductor devices 503 and 603, sensing surfaces 506 and 606, and sensor reservoirs 509 and 609 may be substantially similar or the same as semiconductor device 209, sensing surface 233, and sensing reservoir 223 (FIG. 2), respectively.

[0055] 5 has a semiconductor substrate 504 including an emitter region, a base region, and a collector region. A conductive contact 507 can connect the base region of the substrate 504 to a sensing surface 506, which can be immersed in an analyte solution in a sensor reservoir 509. In other embodiments (not shown), a conductive contact 507 can provide the sensing surface, with at least a portion of the contact immersed in the sensor reservoir 509.

[0056] For example, the contact 507 can be an extended base component that includes a conductive surface. A first end of the extended base component (sensing surface) can be immersed in the analyte solution, while a second end is connected to the base region of the substrate 504. In some embodiments, the sensing surface 506 and / or the conductive contact 507 can be a titanium nitride (TiN) thin film. For example, the extended base component 506 / 507 can include a TiN thin film deposited on a tungsten or any other conductive wire. In some embodiments, the TiN thin film can be sputter deposited on the wire to a thickness of about 100 nm. The sensor reservoir 509 can be directly over the entire base region of the substrate 504, or the sensing surface 506 can be connected to the base region by the extended base component / conductive contact 507.

[0057] A reference electrode 508 (e.g., an Ag / AgCl reference electrode) is also in contact with the analyte solution in the sensor reservoir 509. The sensing signal (collector current I c ) is the base voltage V B and collector voltage V C Zero while the applied emitter voltage V (negative polarity for NPN BJTs and positive polarity for PNP BJTs) E The voltage at the sensing surface 506 can be measured by varying the voltage at the sensing surface 506. However, any suitable voltage can be applied (e.g., from −2 V to 2 V). A response event induced by the presence of the divided reporters in the sensor reservoir 509 generates a collector current Ic can cause a detectable change in

[0058] Proceeding now to FIG. 6, the FET device 613 has a semiconductor substrate 614 that includes three terminals. The three terminals are known as a drain region, a source region, and a gate structure 617. The gate structure 617 may include a gate dielectric (white) over the channel region (between the source and drain) of the substrate 614. The gate structure 617 further includes a gate conductor (gray) that can act as a sensing surface 616 when immersed in an analyte solution in a sensor reservoir 619. In some embodiments, a larger or smaller portion of the gate conductor is immersed to provide the sensing surface 616. The sensor reservoir 619 may be directly over the entire gate conductor, or the sensing surface 616 may be connected to the gate structure 617 by an extended gate conductor. In some embodiments, the sensing surface of the FET device 613 may be a thin metal film such as gold, silver, or copper (e.g., having a thickness of 50 nm to 150 nm).

[0059] The FET device 613 detects a drain current I as a sensing signal by applying a voltage across the drain and source regions of the substrate 614 and a reference electrode 618 in contact with the analyte solution. d For example, the voltages applied to the drain, source, and reference electrodes can be about |25| mV, 0 V, and 0 V to |1 V|, respectively. However, any suitable voltages can be applied, as will be appreciated by those skilled in the art. A response event induced by the presence of the segmented reporters in the sensor reservoir 619 causes a drain current I d detectable changes may occur.

[0060] FIG. 7 is a schematic diagram illustrating a biosensor structure 700 including multiple microfluidic systems on a substrate, according to some embodiments of the present disclosure. The structure 700 has a substrate 709 including wells 703A and 703B (collectively 703). The wells 703 can be microscale or nanoscale structures. For example, the wells 703 can have a diameter and width of about 10 nm to 500 nm, about 500 nm to 100 μm, etc. In some embodiments, the wells 703 can be larger than 100 μm, such as in a macroscale biosensor. Sensing surfaces 706A and 706B (collectively 706) are embedded in the wells 703A and 703B, respectively. The sensing surface 706 is electrically connected to a semiconductor device (see FIGS. 5 and 6). The wells 703 further include a reference electrode (not shown) for applying a voltage to the analyte. Although not shown in FIG. 7, additional components such as channels, reservoirs, metal traces, fluidic gates, etc., can also be within the wells 703. For example, each well 703 can act as an individual laboratory device or reactor and can include components such as those described for biosensors 100 and 200 in FIGS.

[0061] Chemical and / or biochemical changes can be monitored at the sensing surface 706 using multiple wells 703. Each well 703 can be an isolated environment, allowing multiple independent labware to be simultaneously monitored and cross-referenced on a single chip. In some embodiments, the substrate 709 has three or more wells 703 and sensing surfaces 706 (see, e.g., Figures 8(A)-8(C)). Each system / well 703 can be considered a free-standing labware. The wells 703 can optionally be backsealed or oversealed with an immiscible phase or sealant to encapsulate and isolate the contents of each cell 703.

[0062] 8(A)-8(C) are schematic diagrams illustrating structures 800, 810, and 820 including wells of a battery, according to some embodiments of the present disclosure. Structures 800, 810, and 820 each include multiple wells in / on substrates 803, 813, and 823, respectively. The wells may be substantially similar to well 703 shown in FIG. 7 and may include biosensor components as discussed in more detail above. The flow direction of fluids (e.g., samples, analytes, etc.) flowing into structures 800, 810, and 820 is indicated by block arrows.

[0063] Figure 8(A) shows a cross-sectional view of a structure 800 having a substrate 803 containing embedded battery wells 809A-C (collectively 809) and an upper layer channel 806. The lines / curved arrows represent the flow direction of fluid from channel 806 into well 809. Although not shown in Figure 8(A), well 809 includes a sensing surface that is substantially similar to sensing surface 706 shown in Figure 7.

[0064] FIG. 8(B) shows a top view of a structure 810 having a chip substrate 813 including linear channels 816 and battery wells, according to some embodiments of the present disclosure. FIG. 8(C) shows a top view of a structure 820 having a chip substrate 823 including an area array of channels 826A-C (collectively 826) and wells. In FIG. 8(C), "channels 826" refers to both horizontal (inflow direction) and vertical (perpendicular to inflow) channels. The wells (white circles) and sensing surfaces (gray rectangles) shown in FIGS. 8(B) and 8(C) may be substantially similar to the wells 703 and sensing surface 706 shown in FIG. 7.

[0065] In some embodiments, multiple systems / wells integrated into a chip (e.g., substrate 813 or 823) can act as sensing "pixels" for a single use. Multiple pixels (sensing surfaces) can be activated to enable simultaneous monitoring of multiple events, monitoring of spatially coordinated events, time-dependent monitoring of external environmental events, etc. In some embodiments, the wells of the battery can be used to form a stacked (3-dimensional) fluidic network that can simultaneously feed multiple sensing devices in parallel. This can enable silicon-based microfluidic technologies such as multiplexed composite assessment for healthcare with multiple biomarker targets in a single fluid sample.

[0066] The processes discussed herein and their accompanying drawings should not be construed as limiting. One skilled in the art would understand that a variety of techniques may be used in which conditions, components, methods, etc. are varied, ultimately resulting in a biomolecule sensing semiconductor device. Furthermore, conditions may be optionally altered during the process. Furthermore, in some embodiments, processes may be added, omitted, or performed in an alternative order while still remaining within the scope of the present disclosure, as would be understood by one skilled in the art. It should further be noted that a process may be performed by a single entity or by multiple entities. For example, a first entity may manufacture a biosensor device and a second entity may use the device to test a sample.

[0067] The description of various embodiments of the present disclosure is presented for illustrative purposes, but is not intended to be exhaustive and is limited to the described embodiments. Many modifications and variations that do not depart from the scope and spirit of the described embodiments will be apparent to those skilled in the art. The terms used in this specification are selected to best explain the principles of the embodiments, practical applications, or technical improvements over the technology found in the market, or to enable those skilled in the art to understand the embodiments described in this specification.

Claims

1. a component for receiving a sample fluid; Components for preparing the sample fluid; a CRISPR system for splitting reporter species when the sample fluid contains a sequence of interest; a sorting component for separating the resolved reporter species from the sample fluid and CRISPR products; a cavity for receiving an analyte solution containing the resolved reporter species from the sorting component; a sensing surface within the cavity, the sensing surface detecting an electrical signal induced by a reaction event in the analyte solution; a semiconductor device electrically connected to the sensing surface.

2. Each reporter species is A nucleic acid, a reporter ligand attached to a first end of the nucleic acid; and a bead attached to the second end of the nucleic acid.

3. The structure of claim 2 , wherein the screening component comprises a filter having a pore diameter smaller than the beads.

4. The structure of claim 2 or 3, wherein the beads are magnetic particles and the sorting component comprises a magnet.

5. 5. A structure according to claim 1, wherein the reaction event comprises chemical bond formation between the reporter species and a molecule coating the sensing surface.

6. The structure of claim 1 , further comprising a microfluidic channel.

7. 7. The structure of claim 1, wherein the semiconductor device is selected from the group consisting of a field effect transistor (FET) device and a bipolar junction transistor (BJT) device.

8. The structure of claim 1 , wherein the sorting component comprises a deterministic lateral displacement array.

9. A semiconductor device; A silicon substrate containing one or more microfluidic systems, each microfluidic system comprising: an inlet through which a sample fluid can flow; a cavity containing a reporter species and a CRISPR system that splits the reporter species when a sequence of interest is present in the sample fluid; a component for separating the resolved reporter species into an analyte solution; a sensing surface electrically connected to the semiconductor device; and a silicon substrate having a reservoir within which the analyte solution contacts the sensing surface, the reservoir containing components for inducing a reaction event in the presence of the resolved reporter species.

10. 10. The apparatus of claim 9, wherein the semiconductor device is selected from the group consisting of a field effect transistor (FET) device and a bipolar junction transistor (BJT) device.

11. 11. The apparatus of claim 9 or 10, wherein the semiconductor device is a nanowire device.

12. 12. The apparatus of claim 9, wherein the silicon substrate comprises a plurality of the one or more microfluidic systems in respective wells.

13. 13. Apparatus according to any one of claims 9 to 12, wherein each microfluidic system further comprises a fluid gate controlled by an embedded electrode.

14. Providing one or more microfluidic systems on a silicon substrate, each microfluidic system comprising: a component for receiving a sample fluid; Components for preparing the sample fluid; a CRISPR system for splitting reporter species when the sample fluid contains a sequence of interest; a sorting component for separating the resolved reporter species from the sample fluid and CRISPR products; a cavity for receiving an analyte solution containing the resolved reporter species from the sorting component; providing one or more microfluidic systems having a sensing surface within the cavity, the sensing surface detecting an electrical signal induced by a reaction event in the analyte solution; providing a semiconductor device electrically connected to the sensing surface.

15. Each reporter species is A nucleic acid, a reporter ligand attached to a first end of the nucleic acid; and a bead attached to the second end of the nucleic acid.

16. The method of claim 14 or 15, wherein each microfluidic system further comprises a fluid gate controlled by an embedded electrode.

17. 17. The method of any one of claims 14 to 16, wherein the silicon substrate comprises multiple microfluidic systems of the one or more microfluidic systems in individual wells.

18. 18. The method of any one of claims 14 to 17, wherein the semiconductor device is selected from the group consisting of a field effect transistor (FET) device and a bipolar junction transistor (BJT) device.

19. 19. A method according to any one of claims 14 to 18, wherein the cavity has a reservoir, the sample solution contacts the sensing surface within the reservoir, and the reservoir contains a component for inducing the reaction event in the presence of the resolved reporter species.

20. 20. The method of any one of claims 14-19, wherein providing the one or more microfluidic systems comprises preloading a CRISPR reagent into a sample preparation cavity.

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