Patent Application
20240337655
The disclosed technology is generally directed to biosensing articles. More particularly the technology is directed to biosensing articles that provide a luminescent signal.
Sensing devices that can identify biomarkers at low concentration levels are needed in public spaces, personal care items, and especially in clinical environs. Biosensors often rely on complex transduction mechanisms, however robust detection mechanisms are needed. Refined methods based on biochemical transduction agents such as RNA or antibodies have proven effective, however the processing and shelf-stability of these formats are often encumbered by challenges associated with sample treatment and logistics of test result readout. New sensors should have minimal sample treatment requirements, easy-to-read results, as well as shelf-stability, portability, and modular stability of the biochemical components. Sensor stability is especially needed for extended periods in the various conditions encountered during manufacturing, storage, and transportation, such as exposure to high/low temperatures, temperature fluctuations, and humidity.
De novo designed protein switches are powerful tools for specifically and sensitively detecting diverse targets with simple chemiluminescent readouts. Finding an appropriate material host to entrain these protein switches without altering their thermodynamics enables a variety of sensing formats to monitor exposure to pathogens, toxins, or for disease diagnosis. Disclosed herein is a de novo protein-biopolymer hybrid that maintains the detection capabilities induced by the structural change of the incorporated proteins in response to analytes of interest in multiple, shelf-stable material formats. A set of functional demonstrator devices including personal protective equipment such as masks and laboratory gloves, free-standing films, air quality monitors, and wearable devices are presented to illustrate the versatility of the approach. Such formats are designed to be responsive to human epidermal growth factor receptor (HER2), Hepatitis B (HBV), Botulinum neurotoxin B (BoNT/B), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), This rationally designed combination of form and function offers wide opportunities for ubiquitous sensing in multiple environments by enabling a large class of bio-responsive interfaces of broad utility.
Accordingly, aspects of the present invention provide for a solid form sensing article comprising one or more silk fibroin matrix sensing units, wherein each of the one or more silk fibroin matrix sensing units comprises a silk fibroin matrix having entrained therein a plurality of Cage proteins and a plurality of Key proteins which form part of a Cage-Key protein system. Each of the one or more silk fibroin matrix sensing units has a solid form sensing property as measured when exposing the respective silk fibroin matrix sensing unit to an analyte of interest. The plurality of Cage proteins and the plurality of Key proteins have a spatial distribution within the silk fibroin matrix that is selected from the group consisting of homogeneous distribution, random distribution, pseudo-random distribution, and combinations thereof.
Another aspect provided herein is a method of using the solid form sensing article, where the method comprises monitoring a Cage-Key signal associated with the presence of the analyte of interest.
Another aspect provided herein is a method of making a solid form sensing article comprising: 1) drop casting liquid silk fibroin-key/cage matrices converted into sensitive films after water evaporation; 2) lyophilizing silk fibroin-key/cage matrices to make sponges; 3) electrospinning silk fibroin-key/cage matrices to make non-woven mats; and/or 4) inkjet printing and dispensing pico and nanodrops of silk fibroin-key/cage matrices via digitally controlled interfaces on different substrates (including stretchable and flexible fabrics, rigid polymeric surfaces and common objects) to generate a variety of printed patterns.
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
Before example embodiments of an apparatus in accordance with the disclosure are described in further detail, it is to be understood that the disclosure is not limited to the particular aspects described. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. The scope of an invention described in this disclosure will be limited only by the claims. As used herein, the singular forms “a”, “an”, and “the” include plural aspects unless the context clearly dictates otherwise. It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising”, “including”, or “having” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Aspects referenced as “comprising”, “including”, or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements, unless the context clearly dictates otherwise. It should be appreciated that aspects of the disclosure that are described with respect to a system are applicable to the methods, and vice versa, unless the context explicitly dictates otherwise. Numeric ranges disclosed herein are inclusive of their endpoints. For example, a numeric range of between 1 and 10 includes the values 1 and 10. When a series of numeric ranges are disclosed for a given value, this disclosure expressly contemplates ranges including all combinations of the upper and lower bounds of those ranges. For example, a numeric range of between 1 and 10 or between 2 and 9 is intended to include the numeric ranges of between 1 and 9 and between 2 and 10.
De novo designed protein-based biosensors have been recently demonstrated to be a versatile, reconfigurable biochemical transduction mechanism for sensing applications. In these systems, the sensing function is provided by the synergy of two designed protein components, a lucCage (hereinafter “a Cage protein”) and a lucKey (hereinafter “a Key protein”). The Cage protein includes an analyte binding domain. The analyte-binding domain can be de novo designed to specifically bind to an analyte of interest, thus allowing the Cage-Key system to be tuned to the analyte of interest. Proteins can be designed de novo to bind to a target protein with high specificity and affinity by using computational algorithms that have access to an exponentially larger protein sequence space than what is available in Nature. In contrast with naturally occurring proteins, which can bind a limited repertoire of molecules, de novo protein design enables the production of a wide variety of biosensors for any analytes with known structure.
When an analyte of interest interacts with the analyte binding domain, the Cage protein undergoes a conformational change from a closed state to an open state. In the open state, the Key protein forms a complex with the Cage protein. The Key protein includes a key peptide sequence that binds to the open state of the Cage protein and includes a complementary split luciferase fragment. The association of the Key protein and the Cage protein results in reconstitution of luciferase activity. The ensuing luciferase bioluminescence provides a rapid, specific, and sensitive readout of the analyte-driven Cage-Key protein system as a protein switch.
Disclosed herein are bio-responsive sensing materials of broad utility, described in more detail below and shown in
According to an aspect as disclosed herein, the analyte of interest can be the receptor-binding domain (RBD) in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein. In an aspect, the analyte of interest can be the Human Epidermal Growth Factor Receptor 2 (HER2). In another aspect, the analyte of interest can be the Botulinum Neurotoxin B (BoNT/B). In a further aspect, the analyte of interest can be the Anti-Hepatitis B Virus antibody (anti-HBV). In further aspects, the analyte of interest can be cortisol or amylase.
When the Cage-Key protein system is expressed by or contained within a cell, the Cage-Key protein system has an intracellular sensing property. The intracellular sensing property can be measured when exposing the cell expressing or containing the Cage-Key protein system to the analyte of interest. The intracellular sensing property can be a luminescent signal. The intensity of the luminescent signal can be related to the concentration of the Cage-Key protein system, the concentration of the analyte of interest, or both. The intracellular sensing property can indicate the sensitivity of the Cage-Key protein system to the analyte of interest when the Cage-Key protein system is contained within a cell.
When the Cage-Key protein system is dissolved in a solution, the Cage-Key protein system has a solution-phase sensing property. The solution-phase sensing property can be measured when exposing the solution to the analyte of interest. The solution-phase sensing property can be a luminescent signal. In the absence of the analyte of interest a small basal luminescent signal is obtained. When used with furimazine, a substrate, Cage-Key protein system produces a high-intensity luminescence when the analyte is present. This signal increases proportionally to the concentration of analyte of interest bound Cage-Key protein system. The solution-phase sensing property can indicate the sensitivity of the Cage-Key protein system to the analyte of interest when the Cage-Key protein system is dissolved. Alternatively, and additionally, furimazine analogues can be used to produce the luminescent signal, for example, hydrofurimazine, fluorofurimazine, or coelenterazine (CTZ). When the Cage and Key components are present at 0.1 or 1 nanomolar, the detectable range of analyte spans from 10 picomolar to 10 nanomolar or higher.
Despite the higher intrinsic stability of de novo protein switches such as the Key-Cage protein system in comparison with natural proteins, they are commonly stored in refrigerated conditions after snap-freezing or in lyophilized formats. Lyophilization within a stabilizing matrix (such as sugars or polyols) is a common method used to increase the thermal stability of labile molecules by reducing their molecular mobility and degradation processes.
According to an aspect herein, the plurality of Key proteins and the plurality of Cage proteins can be entrained within a matrix. Generally, a plurality of Key proteins, a plurality of Cage proteins, or a plurality of Key-Cage protein systems can be combined with a biopolymer to make a protein-biopolymer hybrid. In a particular example, the matrix can be a silk fibroin biopolymer derived from the native Bombyx mori silkworm fibers. Silk fibroin is a promising matrix for de novo protein switches due to its aqueous solubility, non-toxicity, green chemistry, and polymorphic features.
As shown in
The Cage proteins and the Key proteins can be present in the silk fibroin matrix in a ratio of: 0.13-1.3% w/w for the Cage protein, 0.082-0.82% w/w for the Key protein, 22.5-80% w/w for the silk fibroin considering the total weight of the initial mix where the remainder of the solution is water or water-based, such as a buffer.
The solutions containing the silk fibroin and the plurality of Key proteins and the plurality of Cage proteins can be used to generate a silk fibroin matrix. The distribution of the plurality of Cage proteins and the plurality of Key proteins in the silk fibroin matrix can be a homogeneous distribution, a random distribution, a pseudo-random distribution, or combinations thereof.
In another example, the matrix can include a paper substrate, where the plurality of Key proteins and the plurality of Cage proteins are entrained within pulp or fibers of the paper substrate. In yet another example, the matrix can include sugars or polyols.
The silk fibroin matrix can be associated with a functional material or a material format as illustrated in
The material format can be a three-dimensional porous matrix having a high surface area-to-volume ratio, such as a sponge or a foam. The material format can be made as a sponge by snap-freezing and lyophilizing solutions containing silk fibroin, Key proteins and Cage proteins. The material format can be a high surface-to-volume porous formats to sample air as a bioresponsive bulk material. For example, the material format can be an air filter.
The material format can be a non-woven membrane, a mesh, or combinations thereof. More specifically, the material format can be an electrospun mat. This material format can serve both for detection and small-pore size filtration by tuning the electrospinning parameters, with end utility in applications such as functional face coverings or air filters for simultaneous protection and monitoring of biological contamination in aerosols.
The material format can be a printable ink. The amphiphilic nature of the protein chains of silk fibroin allows for the generation of pico- to nanoliter solution drops of silk, enabling use in printing technologies. The rheological properties of the inks, such as viscosity and surface tension, do not need to be modified with non-compatible organic or inorganic surfactants, which could negatively interact with doping agents, therefore enabling the functionalization of any substrate of interest.
Printing can provide a modular approach for the use of multiple Key-Cage systems across a variety of surfaces, adding utility to wearable interfaces, surfaces, containers, or other high-touch surfaces providing early warning against exposure to hazardous agents. To generate a printable ink, a solution of silk fibroin can be combined with Key proteins and Cage proteins and additives, for example, Tween. The printable ink can be deposited as spots on a surface, where the spots are arranged singly or printed in a pattern.
Water-based suspensions of silk fibroin can be transformed into a variety of other material formats, including hydrogels, microneedles, and other responsive biointerfaces. These material formats can allow the silk fibroin matrix to be shelf-stable and can be stored without refrigeration while preserving the sensitive detection capabilities of the Key-Cage protein system, as will be discussed later.
The material format can be formed according to the following methods starting from matrix solutions containing Key proteins, Cage proteins, and silk fibroin. In an example, the matrix solution containing Key proteins, Cage proteins, and silk fibroin can be drop-cast and converted into sensitive films after water evaporation. Free-standing films can be formed by casting matrix solutions containing silk fibroin, Key proteins, and Cage proteins on a PDMS substrate and allowing the water to evaporate overnight, leaving a film.
In another example the matrix solution containing Key proteins, Cage proteins, and silk fibroin can be lyophilized to make porous items such as sponges, as shown in
In yet another example, matrix solutions containing Key proteins, Cage proteins, and silk fibroin can be electrospun to make non-woven mats. The electrospun mat can be generated by preparing a matrix solution of silk fibroin, Key proteins and Cage proteins, and an additive such as polyethylene glycol, and extruding through a needle having an applied voltage at a flow rate where a grounded fiber collector is positioned away from the needle. The sensing capability of silk fibroin matrix on porous substrates can be improved by fabricating non-woven meshes through direct electrospinning of the silk fibroin matrix.
Bioluminescence imaging can be particularly suited for biomarker detection outside laboratory setting given its sensitivity and ease of use. As shown in
Addition of furimazine to analyte-bound Key-Cage protein complex results in luminescence, a signal that can be readily observed and measured by a detector. The readout can be easily performed by using low-cost digital cameras and/or smartphones. Although the addition of furimazine is required and the luminescence measurement should be performed in a dark environment, both of these requirements are easily achievable. For example, a kit can be provided that includes furimazine and an app or program that can be integrated with digital cameras or smart phones to facilitate sample processing. The kit can further include an enclosure or covering to assist the user in providing a darkened environment for the readout. Additionally, the kit can include standard solutions for comparison.
When entrained in the silk fibroin matrix, the plurality of Cage proteins and the plurality of Key proteins are not contained, as within a cell, and are not free, as in a solution. When the plurality of Key proteins and the plurality of Cage proteins are entrained in the silk fibroin matrix, the silk fibroin matrix has a silk fibroin matrix sensing property. The silk fibroin matrix sensing property can be a luminescent signal. The intensity of the luminescent signal can be related to the concentration of analyte of interest. The silk fibroin matrix sensing property can be different from one or both of the intracellular sensing property and the solution-phase sensing property. The silk fibroin matrix sensing property can indicate the sensitivity of the Cage-Key protein system to the analyte of interest when the Cage-Key protein system is entrained in the silk fibroin matrix. Remarkably, the silk fibroin matrix sensing property is highly sensitive to the presence of the analyte of interest. This is a surprising result because the mobility of the Key proteins and Cage proteins is decreased when they are entrained in the silk fibroin matrix. This finding enables new sensing applications using new solid form factors (e.g., environmental passive monitoring) that would not be possible with a solution-based system. The de novo proteins are engineered in the lab and are based on a thermodynamic model to allow for their folding/unfolding behavior. It is not obvious that the de novo proteins will maintain their function when they are mixed in a proteinaceous matrix such as silk (i.e. mixing what doesn't exist in nature with what is derived from a “caterpillar”). The folding/unfolding behavior (i.e., the conformational change of the de novo proteins) is an essential feature that allows de novo protein switches to respond to external stimuli (e.g., the presence of markers, toxins, and other contaminants). The preservation of their ability to fold/unfold after the inclusion in a solid silk matrix is the key to the functionality of the sensing system.
Free-standing bioresponsive films and sponges can be fabricated by adjusting the ratio of Key proteins and Cage proteins within the silk fibroin solution to tune material assembly, performance, and permeability. The viability of the de novo designed Key-Cage protein system contained in the solid material formats can be assessed by dissolving the solid material format, such as sponge or film formats, in water and then exposing them to various concentrations of the analyte of interest to evaluate the protein performance through its chemiluminescent response (
Evaluation of the luminescent signal shows the Key-Cage protein system is responsive and can detect the analyte of interest within minutes (˜10-20 minutes). The Limit of Detection (LOD) for the analyte of interest using can be between nM to pM using reconstituted Key proteins and Cage proteins from material formats such as sponge or film. For example, HER2, BoNT/B, and anti-HBV are detected at nM levels (See
To utilize the sensing ability or analyte responsiveness of the Key-Cage protein system in specific areas, the silk fibroin matrix can be incorporated within a solid form sensing article. Examples of solid form sensing articles include a breast pad, a jar lid, a mask, a glove, and an aerial drone as shown in
At least one silk fibroin matrix sensing unit can be incorporated in the solid form sensing article. The silk fibroin matrix sensing unit can make up the entirety of the solid form sensing article. Additionally and alternatively, multiple silk fibroin matrix sensing units can be incorporated in the solid form sensing article. The solid form sensing article can include one or more silk fibroin matrix sensing units. The solid form sensing article can include two, three, four, five, six, seven, eight, nine, or ten or more silk fibroin matrix sensing units. The solid form sensing article can include ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, or one hundred or more silk fibroin matrix sensing units. The solid form sensing article can have two or more silk fibroin matrix sensing units where at least two of the two or more silk fibroin matrix sensing units have different Cage-Key protein systems tuned to different analytes of interest. The solid form sensing article can have twenty-five, thirty, forty, fifty, sixty, seventy, eighty, ninety, or one hundred or more silk fibroin sensing units, where at least twenty-five, thirty, forty, fifty, sixty, seventy, eighty, ninety, or one hundred have different Cage-Key protein systems tuned to different analytes of interest.
The material format of the silk fibroin matrix and the concentration and distribution of the plurality of Cage proteins and the plurality of Key proteins within the silk fibroin matrix provide a solid form sensing property. When the solid form sensing article is exposed to the analyte of interest, the solid form sensing property of the solid form sensing article is activated. The solid form sensing article can thus be used to detect at least one analyte of interest.
The solid form sensing article can be an item that can be used in or placed in an area where the analyte of interest may be present such as a high-touch surface. In some examples, the solid form sensing article can be a mat, a pin, a sticker, a utensil, or a magnet where the silk fibroin matrix sensing units can be printed on the surface. In other examples, the solid form sensing article can be a type of personal protective equipment such as a glove, a face mask, or an article of clothing. The material format can be a printable ink such that silk fibroin matrix sensing units can be printed on a surface, such as on a glove or mask as shown in
Additionally, or alternatively, the solid form sensing article can be a free-standing film. The silk fibroin matrix sensing units can be incorporated in the solid form sensing article as solid components. The solid form sensing article can be used for routine monitoring of specific biomarkers, for example with point-of-care devices. In another aspect, the solid form sensing article can be a porous material such as a filter through which air is circulated.
The solid form sensing article can be a personal hygiene item having one or more silk fibroin matrix sensing units. Examples of solid form sensing articles in personal hygiene include disposable wipes, sanitary pads, and dental floss. Generally, the one or more silk fibroin matrix sensing units can be positioned to optimize contact with bodily fluids potentially containing an analyte of interest. In a particular example the solid form sensing article can be a breast pad as shown in
In another aspect, the solid form sensing article can be a part of an air quality monitoring system for detecting airborne analytes and routine monitoring. In another aspect, the solid form sensing article can be a porous material such as a filter through which air is circulated. In another aspect, the solid form sensing article can be a material exposed to air.
In one example, the solid form sensing article can be incorporated in an aerial drone. The silk fibroin matrix sensing units can be components lodged on the exterior of the aerial drone for exposure to the air during flight. In one example, a viral-sensing aerial drone was generated which was obtained by freeze-drying the silk fibroin matrix in the shape of the top half of the ˜3 cm aircraft (
In yet another aspect, the solid form sensing article can be a part of a food quality monitoring system for detecting food-borne analytes. The solid form sensing article can be incorporated in a container or a cover for a container. In an example, the silk fibroin matrix sensing units can be printed on the inside of a lid of a jar of food, as shown in
The solid form sensing article can be a wearable device or an item of jewelry such as a bracelet, a pin, an earring, or a pendant. The wearable device or item of jewelry can include at least one silk fibroin matrix sensing unit for detecting an analyte of interest in the environment of the user. Alternatively, the wearable device or item of jewelry can include at least one silk fibroin matrix sensing unit for detecting an analyte of interest originating from the user.
In use, the solid form sensing article can be directly exposed to the analyte. More specifically, the material format including the silk fibroin sensing units can be exposed to the analyte of interest such that the analyte contacts the silk fibroin sensing units. After exposure, the analyte can be lodged on the surface of the material format. The analyte can penetrate the pores of the material format, for example in the case of sponges. The analyte can adhere to fibers, for example in the case of mesh or electrospun mats.
Following exposure to the analyte, at least a portion of the material format containing the silk fibroin sensing units can be dissolved to form an analyte solution. The analyte solution contains silk fibroin, Key proteins, Cage proteins, Key-Cage protein complex, and the analyte. These components are released from the silk fibroin matrix upon dissolution. In the analyte solution, the Key proteins, Cage proteins, and the analyte are mobile and free to form the Key-Cage protein complex with the analyte of interest. The analyte solution is exposed to furimazine such that the Key-Cage protein complex generates a chemiluminescent signal that can be detected.
In one example, the film containing Key proteins and Cage proteins can be partially hydrated with contaminated biological fluids or deionized water that is spiked with the target of interest (mimicking contaminated biological fluids). This step of partial hydration favors the interaction between the analyte and the Key proteins and Cage proteins that are far from the film surface. This approach can provide advantages for use outside the laboratory and does not require complete dissolution of the sensing substrate, which can facilitate usage by untrained users while providing quantitative information on the sensing substrate within minutes.
Additionally, and alternatively, the material format can be used for sensing without complete dissolution of the material format. For example, the material format including the silk fibroin sensing units can be exposed to the analyte of interest, directly exposed to furimazine, and the luminescent signal detected as emanating from the material format. As shown in
Examples of this method of use are further illustrated in
In one example the solid form sensing articles are laboratory gloves and surgical masks. In this case the material format is a printable ink, and silk fibroin sensing units sensitive to RBD were printed on the outer surface of the laboratory gloves and exterior surface of a surgical mask. In another embodiment, the silk fibroin sensing units can be printed on the interior surface of a surgical mask. After exposure to RBD, the gloves and mask show a chemiluminescent response proportional to the concentration of the analyte while maintaining the geometry of the printed pattern. The printed patterns provide a luminescent response that is directly proportional to the analyte concentration (
The surgical mask having sensing units provides a convenient complementary approach to nasopharyngeal swabs for the detection of SARS-COV-2 and was evaluated by spraying furimazine and RBD target to mimic the breathing of potentially infected users (
In another example, the solid form sensing article is a silicone breast pad. This solid form sensing article was selected given the importance of HER-2 as a breast cancer biomarker in nipple fluids and can serve as a potential interface for early detection of breast cancer, to assess the efficacy of treatment, or to discriminate between benign or malignant discharge. HER2 concentration is tracked directly on the dotted pattern dispensed on the silicone pad in a short timeframe (˜20 minutes) and within the nM range (
In yet another example, the detection of toxins relevant to food safety and human health such as botulin were also explored by using relevant de novo Key-Cage protein systems in a matrix of silk fibroin as a printable ink and printed as patterns on the inside of a food jar lid. As in the case described above, this approach could allow for the detection of contamination, spoilage, or tampering of the contents by examination of the lid's luminescence response in 20 minutes and at the toxin concentration within the nM range (
Biosensors are prone to interference given the large amounts of non-specific interactions with background molecules, which can affect their sensing capabilities in biofluids. To further evaluate the response of the silk fibroin matrix sensing units to different specimens, simulated saliva spiked with different concentrations of RBD was used to test the performance of a solid form sensing article (a responsive film) in complex media. RBD was detected in simulated saliva down to 10 pM concentrations without compromising the dynamic sensing range (
Additionally, the specificity of RBD bioresponsive films was evaluated by testing their responsiveness to potentially interfering analytes (
Although the Key-Cage protein systems can be stored in refrigerated conditions at −80° C., the integration in the silk fibroin matrix can preserve the sensing performance (
De novo designed protein switches such as the Key-Cage protein system are powerful tools to specifically and sensitively detect diverse targets with simple luminescent readouts. Finding an appropriate material host for de novo design protein switches without altering their thermodynamics while preserving their intrinsic stability over time enables the development of a variety of sensing formats to monitor exposure to pathogens, toxins, and for disease diagnosis. Silk fibroin is an attractive matrix for lyophilization to obtain stable silk fibroin matrices that can be quickly (within seconds) dissolved for readout. Additionally, silk provides material versatility, including the ability to cast films which reduces the cost of fabrication and processing time and results in shelf-stable formats that can be easily handled and shipped for analysis in the field.
Silk fibroin is advantageous because of its polymorphism, which allows for processing and reshaping into different material composites that preserve the biological function of their constituents.
The demonstrations included herein show the interplay between form and function, where the different bioresponsive formats can be directed to different end uses. The different formats can be tuned for optimal analyte diffusion and permeability by control of the directed assembly of the silk fibroin matrix through the modulation of its inter- to intramolecular bond ratios for efficient analyte collection, sampling, and sensing. This versatility ultimately can be exploited to optimize sensor performance and use in a variety of settings, from the laboratory to the clinic, to the home.
Disclosed herein is a de novo protein-biopolymer hybrid that maintains the detection capabilities induced by the conformational change of the incorporated proteins in response to analytes of interest in multiple, shelf-stable material formats without the need of refrigerated storage conditions. A set of functional demonstrator devices including personal protective equipment such as masks and laboratory gloves, free-standing films, air quality monitors, and wearable devices is presented to illustrate the versatility of the approach. Such formats are designed to be responsive to human epidermal growth factor receptor (HER2), anti-hepatitis B (HBV) antibodies, Botulinum neurotoxin B (BoNT/B), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This combination of form and function offers wide opportunities for ubiquitous sensing in multiple environments by enabling a large class of bio-responsive interfaces of broad utility.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.
Sodium carbonate, lithium bromide, and 1× phosphate buffer (PBS) were purchased from Sigma-Aldrich (USA). Ethanol (100%) and Teknova 10% TWEEN-20 solution were purchased from Fisher Scientific. The furimazine substrate was part of the kit Nano-Glow® Luciferase Assay System and was purchased from Promega. Human HER2/ErbB2 Protein, His Tag (MALS verified) was purchased from Acro Biosystems. LucKey and lucCage constructs (lucCageRBD, lucCageHer2, lucCageHBVa and lucCageBot) were expressed and purified as previously reported (See: Tao, H. et al. Inkjet Printing of Regenerated Silk Fibroin: From Printable Forms to Printable Functions. Adv Mater 27, 4273-4279 (2015). https://doi.org:10.1002/adma.201501425, which is hereby incorporated by reference in its entirety for all purposes). Monomeric SARS-CoV-2 RBD was expressed and purified as previously described (See: Marelli, B. et al. Programming function into mechanical forms by directed assembly of silk bulk materials. Proc Natl Acad Sci USA 114, 451-456 (2017). https://doi.org:10.1073/pnas.1612063114, which is hereby incorporated by reference in its entirety for all purposes). The anti-HBV antibody HzKR127-3.2 was purified as previously described (See: Langan, R. A. et al. De novo design of bioactive protein switches. Nature 572, 205-210 (2019). https://doi.org:10.1038/s41586-019-1432-8; and Quijano-Rubio, A. et al. De novo design of modular and tunable protein biosensors. Nature 591, 482-487 (2021), both of which are hereby incorporated by reference in their entirety for all purposes). Botulinum neurotoxin B HcB expressed as previously reported (See: Matzeu, G. et al. Large-Scale Patterning of Reactive Surfaces for Wearable and Environmentally Deployable Sensors. Adv Mater 32, e2001258 (2020). https://doi.org:10.1002/adma.202001258, which is hereby incorporated by reference in its entirety for all purposes). All chemicals were used as received and they followed trace metal standard, when possible. Silk cocoons from Bombyx Mori silkworm were purchased from Tajima Shoji Co. (Yokohama, Japan). Deionized water with resistivity of 18.2 MΩ cm was obtained with a Milli-Q reagent-grade water system and used to prepare aqueous solutions. Polystyrene sheets (1.6 mm thickness) used as substrates for printing were purchased from McMaster Carr. 96 well plates (Corning® 96 Well Half-Area Microplate, CLS3693) were purchased from Sigma-Aldrich (USA). Surgical masks (Disposable 3-Ply Masks, 12-888-001A), laboratory nitrile gloves (579585XS), and PCR tubes were purchased from Fisher Scientific. Simulated saliva (1700-0305) was purchased from Pickering Laboratories.
Regenerated silk fibroin (RSF) solution was isolated from B. mori cocoons as previously reported (Langan, 2019; Quijano-Rubio, 2021). Briefly, silk cocoons were shredded, separated from impurities, and boiled in a 0.02 M solution of sodium carbonate to remove sericin. Boiling time was set to 120 minutes to fabricate bio-reactive films, sponges, and inks, and to 30 minutes to prepare mixes for electrospinning. The fibers were rinsed with deionized water three times for 20 minutes and air-dried overnight. The dried silk mats were subsequently dissolved in a 9.3 M solution of lithium bromide at 60° C. for 4 hours. The chaotropic salt was then removed through dialysis (dialysis membranes MWCO 35 kDa) against deionized water for 48 hours, changing the water 6 times at regular intervals, which yielded a 7-8 wt % silk solution. The final solution was centrifuged at ˜12,700×g for 20 minutes, filtered to separate remaining impurities, and stored at 4° C.
LucCage and lucKey of desired concentrations were prepared from the initial stock solutions listed in Table 1. Aliquots of the lucCage and lucKey stock solutions were combined with silk solutions at 2 wt % or 4 wt %, to obtain sensing films and sponges with different concentrations, as reported in Table 2. To prepare bio-reactive film formats, 100 μL of the sensing mix was cast on PDMS substrates. Films were dried at room temperature (25° C.) overnight. To prepare bio-reactive sponges, 100 μL of silk-based solution was aliquoted in PCR tubes, flash-frozen, and lyophilized overnight with a Labconco FreeZone 2.5 Liter Benchtop Freeze Dry System (USA). Bio-reactive films and sponges were stored in Eppendorf tubes at room temperature before undergoing further testing.
The lucKey-lucCageRBD stock solutions were diluted to the final concentration of 25 nM in 2 wt % silk fibroin solution using deionized water prefiltered with a 0.22 m syringe filter. The solution was mixed with a plate shaker for 45 minutes to enable pre-equilibration and subsequently 0.2 wt % Tween 20 was added to yield the printing mix. 20 μL of silk ink was printed onto white polystyrene substrates, surgical masks, and nitrile laboratory gloves using an HP D300e Digital Dispenser and T8+ dispense head cassettes. The printing process was influenced by the viscosity of the solution, the surface tension, and the alignment between substrates and dispense head, which affected the dispensing accuracy. Furthermore, the hydrophobicity of the substrates greatly impacted the resolution of the printed patterns. Although different types of surfaces were used, the thickness of the chosen substrates (1.6 mm) enabled high dispensation accuracy and spot alignment during the printing process. The printed patterns were designed with the HP Bio Pattern software. The printing resolution was optimized by tuning the number of layers dispensed and the volumes dispensed per-spot. The final volume of the pattern was given by the total volume dispensed per-spot multiplied by the number of spots in the pattern. 25 nM LucKey-lucCageHER2 and 50 nM BoNT/B solutions were prepared and manually dispensed on a silicone breast pad in 2 ul drops (HER2) or a jar lid in 20 ul (BoNT/B).
Chemiluminescent readings were recorded with a Synergy HT Microplate Reader (BioTek, USA). The sensing silk-based sponges and films were fully dissolved in deionized water and mixed for 45 minutes before analysis. 15 μL of furimazine substrate (Nano-Glo luciferase assay reagent, Promega) was added to the sensing mix to obtain a final total volume of 2015 μL. 95 μL aliquots of the lucKey-lucCage silk solutions were transferred into a white polystyrene 96 well plate and the solution was monitored to obtain the baseline reading by acquiring the bioluminescence signal every 3 minutes for 50 minutes. After this time, 5 μL of serially diluted target proteins were added to the mix into the 96 well plate. The final solution was mixed for 5 minutes. Bioluminescence kinetics were recorded for 3 additional hours to acquire the response of the dissolved bio-reactive silk formats in presence of various analytes concentrations. The limit of detection (LOD) was calculated as the value related to the cross point between the extrapolation of the lines defining the nonresponsive range and linear-response range of the system.
The lucCage biosensor proteins lucCageRBD, lucCageHBVa, lucCageHER2, lucCageBot and lucKey were purified as described. Briefly, glycerol stocks of E. coli Lemo21(DE3) cells (NEB) previously transformed with pET29b+ plasmids encoding the synthesized genes of interest were grown for 24 hours in LB media supplemented with kanamycin. The starter culture was then inoculated at a 1:50 mL ratio in the Studier TBM-5052 autoinduction media supplemented with kanamycin, grown at 37° C. for 2-4 hours, and then grown at 18° C. for an additional 18 h. Cells were harvested by centrifugation at 4000 g at 4° C. for 15 min and resuspended in 30 ml lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.02 mg/mL DNAse). Cell resuspensions were lysed by sonication for 2.5 minutes (5 second cycles). Lysates were clarified by centrifugation at 24,000 g at 4° C. for 20 min and passed through 2 ml of Ni-NTA nickel resin (Qiagen, 30250) pre-equilibrated with wash buffer, (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole). The resin was washed twice with 10 column volumes (CV) of wash buffer, and then eluted with 3 CV of elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). The eluted proteins were concentrated using Ultra-15 Centrifugal Filter Units (Amicon) and further purified by using a Superdex™ 200 Increase 10/300 GL (GE Healthcare) size exclusion column in Tris Buffered Saline (TBS; 25 mM Tris-HCl pH 8.0, 150 mM NaCl). Fractions containing monomeric protein were pooled, concentrated, snap-frozen in liquid nitrogen, and stored at −80° C.
Bioluminescence images were acquired with the Molecular Imaginer® ChemiDoc XRS+Imaging System (Bio-Rad) using the Chemi High Resolution mode. Images were acquired at time intervals of 5 minutes, for a total of 25 minutes.
Imaging of films: For these experiments, RSF films were prepared with the same mix used for printing. Briefly, 20 μL of bio-reactive silk solution at a final concentration of 2 wt % were cast on polystyrene substrates and let dry overnight at room temperature. To analyze the film responsiveness to various target concentrations, films were dissolved in 15 μL of deionized water containing 3% of furimazine substrate. For the experiments with RBD, films were dissolved both in water and in simulated saliva before readouts. After the addition of furimazine, 5 μL of serially diluted target solutions were added to the dissolved films to evaluate the bioluminescence signal for each analyte concentration. Example results are shown in
To check the specificity of the sensing films, interference experiments were carried out using lucCageRBD films. These were exposed to 5 μL of serially diluted RBD target (control) or interfering analytes (BoNT/B, anti-HBV antibody, and HER2 ectodomain protein), and the corresponding bioluminescence signal was detected. The concentration of the serial dilutions of the analytes was set to 0.32, 1.6, 40 and 200 nM. The results are shown in
Imaging of patterned ink (
Imaging of surgical masks (
For an easier visualization, all the collected images were processed in false color (reverse Viridis color map reference) and using image processing libraries in Python software. The reverse Viridis color map associates high luminesce readings to yellow hues and low/absent signals to blue hues. The green gradient that developed between the blue and yellow hues accounts for the growth in chemiluminescence readouts. The false color images were post-analyzed using the software Fiji-ImageJ to quantify and relate the intensity variations in luminescence to variations in analyte concentrations.
The lucKey-lucCageRBD initial stock solutions were diluted in deionized water and mixed for 45 minutes with a plate shaker to enable equilibration. The solution was then combined with a mixture of 7 wt % RSF and 1.2 wt % polyethylene glycol to obtain a final concentration of lucKey-lucCageRBD of 25 nM. A custom-made electrospinning setup was used to fabricate the non-woven mats. Prior to electrospinning, the metal collection circular plate was coated with Teflon spray to facilitate sample removal after electrospinning. 1 mL of solution was then extruded through an 18-gauge needle at a flow rate of 0.90 mL/hr. An applied voltage of 16 kV to the needle caused jet initiation and fiber collection on a grounded collector positioned 18.5 cm away. The electrospinning apparatus was contained in an electrically insulated and environmentally controlled enclosure which was maintained at 25% humidity. After electrospinning, the fiber mat was collected, cut in circles (1 cm of diameter) with a laser cutter (Trotec Speedy 300) and subsequently exposed to target and furimazine before bioluminescence imaging. Results are shown in
To test the ability of the bio-responsive silk sponges to detect the SARS-CoV-2 RBD protein in air, 25 nM lucKey-lucCageRBD silk sponges were exposed to aqueous vapor with and without 500 pM of RBD target protein. The sponges were placed in a stainless-steel cylindrical chamber kept on a heating plate at 120° C. containing a water bath to generate the water vapor and exposed for 20 minutes. To prevent the direct contact of the sponges with the bath, they were placed on a metallic mesh. After the exposure to the vapor, the bioluminescence response of the sponges dissolved in deionized water was acquired for 1 h with a bench top plate reader.
Preliminary studies were carried out to assess the impact of RSF at different concentrations on the lucKey-lucCage protein biosensor functionality and the HER2 protein complex was used as a model. In these experiments, different combinations of lucKey-lucCage at final concentrations of 1 nM and 5 nM were embedded into RSF solutions at 4 wt %, 3 wt %, 2.5 wt %, and 2 wt % (
The lucKey-lucCage biosensing units maintained detecting capabilities after combination with silk fibroin solutions (
The concentration of RSF influenced the luminescence intensity but did not compromise the overall sensing performances in terms of dynamic range and average LODs. The trend shows that the lower the amount of silk (2 wt %), the higher the luminescence intensities were. Luminescence intensities range from ˜1×106 (with silk at 2 wt %) down to ˜2.6×105 counts (with silk at 4 wt %), at high target concentrations (125 nM HER2, 5 nM lucKey-lucCage,
Additional testing was carried out on silk-based solutions at 4 wt %, 3 wt %, 2.5 wt %, and 2 wt % embedding 0.5 nM of lucKey-lucCage (Figure S1A). The sensing response for silk solutions at 4 wt % differs from the other mixtures during the detection of low target concentrations (range 0.04-5 nM). The luminescence intensities decreased (down to 3000-8000 counts) even with the highest target concentration (125 nM). The overall detecting performances of 3 wt %, 2.5 wt %, and 2 wt % (0.5 nM lucKey-lucCage) silk-based solutions were preserved, but luminescence counts were lower (8000-38000 counts).
It is likely that silk fibroin interacts with the lucKey-lucCage protein complex through electrostatic and hydrophobic interactions. These types of interactions are involved in the stabilizing effect of silk towards proteins and enzymes in solution, preventing protein-protein interactions and thus aggregation. In this case, higher concentrations of silk fibroin could shield the analyte binding sites of the lucKey-lucCage complex, leading to a drop in bioluminescence intensity.
After the optimization of the lucKey-lucCage-RSF hybrid in solution, the sensing performances of the complex after incorporation in the solid-state were tested to evaluate the ability of RSF to generate shelf-stable self-standing sensing substrates (
Bioluminescence response showed that the lucKey-lucCage system was stable and responsive both in the sponge (
The chemiluminescent response of 4% and 2% silk sponges (
Different concentrations of lucKey and lucCage (1 nM and 5 nM) were tested to analyze their effect on the overall performance of HER2 and RBD detection in sponges and film formats.
The concentration of lucKey-lucCage did not affect the dynamic sensing range (2.5-125 nM) and LOD, i.e., 1.1±0.1 for lucCageHER2 sponges (
The best performances were achieved with sponges and films prepared with 5 nM lucKey-lucCageRBD and 2 wt % or 4 wt % RSF solution, which enabled accurate detection of RBD within the sensing range of 20-150 pM. For each analyte, the concentration of lucKey-lucCage was adjusted to optimize the sensing performances of the solid substrates.
The performance of the lucKey-lucCageRBD system towards the Omicron variant of the SARS-CoV-2 Spike RBD target was also tested in a 2% RSF solution. Although the system has not been optimized for the detection of Omicron, the lucKey-lucCageRBD system can detect the variant at nM concentration (
To speed up the detection method and facilitate sample analysis in non-laboratory settings, the sensing performances of responsive films were evaluated with a bioluminescence camera. Films were cast on white polystyrene substrates using the approach described in Methods Section 2 and the sensing performances of the films were assessed using a chemiluminescent imaging system (Methods Section 6, and Supplementary Methods 2). This detection method was selected since the reading unit can be potentially miniaturized into a portable device to enable delocalized monitoring outside laboratory-controlled conditions.
Imaging of lucKey-lucCageRBD films was achieved with the lucKey-lucCage complex at a concentration of 25 nM (
To assess the stability of the system in harsh conditions, the lucKey-lucCageRBD was electrospun to obtain a non-woven mat that could have applications in air filtration units. The biosensing mix maintained its responsiveness when integrated in an electrospun mat (
Bio-responsive patterns were printed or deposited onto different interfaces, including, a silicone breast pad (
The printing process did not affect the sensing capability of the lucKey-lucCage complex, and the different printed patterns provided a chemiluminescent response proportional to various target concentrations in presence of furimazine substrate (
The analysis of the mask (
Aiming at the detection of HER2, HER2-RSF hybrid complexes were integrated onto a silicone breast pad. As most breast cancers arise from the ductal epithelium, examination of the breast ductal fluid can be used for early detection of breast cancer and to monitor and predict the efficacy of therapies. HER2 is found in nipple fluids at a higher concentration than in blood, and soluble HER-2 (whose normal concentration is in the range of ng/ml) is present in higher concentrations in nipple fluid of patients with tumors. Thus, sensing breast pads could represent a breakthrough in the early detection of breast cancer when spontaneous nipple discharge is present, as they could detect whether the discharge is benign or malign. HER2-RSF sensing pads were responsive and able to detect to different concentrations of HER2 within minutes (˜20 min) in the nM range (
In summary, the results presented in
Accelerated aging tests for the RBD bio-reactive films and sponges were performed to evaluate the intrinsic stability of the 2 wt % and 4 wt % silk formats embedding lucKey and lucCage at 1 and 5 nM over time (
The luminescence intensity recorded for sponges prepared with RSF at 4 wt %, and lucKey-lucCage at 5 nM decreased over time when using 50 nM RBD target concentration, with a variation of ˜25% between the first (7 hours) and final reading (4 months,
Films at 2 wt % silk, 5 nM lucKey-lucCage analyzed at 150 nM of RBD concentration were found to be more stable, as luminescence intensities varied of ˜4% between the first and the last reading (
Some of the computed LOD values listed in
The stability of HER2-RSF hybrid sponges was re-tested after storage at room temperature (20-25° C.) over 1 year. Despite the lucKey-lucCage sensing components are typically refrigerated at −80° C., the integration in the RSF matrix preserved their sensing capabilities (
To test the ability of the bio-responsive silk sponges to detect the SARS-CoV-2 RBD protein in air, 25 nM lucKey-lucCageRBD silk sponges were exposed to aqueous vapor with and without 500 pM of RBD target protein. The sponges were placed in a stainless-steel cylindrical chamber kept on a heating plate at 120° C. containing a water bath to generate the water vapor and exposed for 20 minutes. To prevent the direct contact of the sponges with the bath, they were placed on a metallic mesh. After the exposure to the vapor, the bioluminescence response of the sponges dissolved in deionized water was acquired for 1 h with a bench top plate reader.
Examples disclosed herein are further described in d'Amone, L., Matzeu, G., Quijano-Rubio, A., Callahan, G. P., Napier, B., Baker, D., & Omenetto, F. G. (2023). Reshaping de Novo Protein Switches into Bioresponsive Materials for Biomarker, Toxin, and Viral Detection. Advanced Materials, 35(11), 2208556 and the accompanying Supporting Information. https://doi.org/10.1002/adma.202208556, both of which are incorporated by reference in their entireties for all purposes.
This application relates to, incorporates by reference for all purposes, and claims priority to U.S. Application Ser. No. 63/488,687, filed Mar. 6, 2023.
This invention was made with government support under grant N00014-19-1-2399 awarded by the Office of Naval Research. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63488687 | Mar 2023 | US |
