A nonperturbing sensor for continuous monitoring of immune biomarkers in biofluids

Abstract

A biosensor for in situ continuous monitoring of molecular biomarkers includes a flexible organic or inorganic layer, and a biorecognition element microarray placed on the organic or inorganic layer. In some aspects, the flexible layer is placed upon a disk that may be made of, for example, one or more of poly dimethylsiloxane (PDMS), silicone, polyurethane, polymers, and hydrogels. The organic or inorganic layer enables the immobilization of biorecognition elements, such as polystyrene (PS) film comprises functional groups from Gamma irradiation to increase biorecognition element binding. The biorecognition elements may include, for example, one or more of an antibody, aptamer, peptide, molecularly imprinted polymers, nucleic acid.

Description

A NONPERTURBING SENSOR FOR CONTINUOUS MONITORING OF IMMUNEBIOMARKERS IN BIOFEUIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority from, and incorporates by reference the entire disclosure of, U.S. Provisional Patent Application No. 63 / 534,746 filed on August 25, 2023.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R21EB029064 and R35GM147568 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates generally to biosensors and more particularly, but not by way of limitation, to a floating biosensor for in situ continuous monitoring of immune biomarkers at attomolar concentrations with 15-minute temporal resolution.BACKGROUND

[0004] This section provides background information to facilitate a better understanding of the various aspects of the disclosure. It should be understood that the statements in this section of this document are to be read in this light, and not as admissions of prior art.

[0005] Continuous and multiplexed quantification of protein biomarkers with low abundance in complex biofluids is important for biomedical research and clinical diagnostics. Emerging amplification technologies based on protein arrays and bead-based immunoassays offer superior sensitivity7and multiplexed capabilities over conventional protein quantification approaches. However, continuous sampling without perturbing the biological systems remains challenging.

[0006] Comprehensive and accurate characterization of complex and non-linear biological systems, such as cell cultures, organoids, tissues, and organs, require quantitative time-varying information of component biomolecules at many different time scales, from milliseconds to days. Sensitive detection and quantification of various protein biomarkers in biological fluids and tissues is of fundamental importance in biomedical research and clinical diagnostics.Conventional techniques such as enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry have enabled the discovery and study of proteins in biological samples. However, these techniques cannot provide in situ analysis without compromising the samples. For example, to monitor and quantify the protein concentration changes over time in cell culture media, sample replicates need to be prepared and characterized at specific time points throughout the duration of a study. The large variations between different batches of cell cultures can potentially mask the temporal concentration changes in the biomarkers of interest. These methods fail to provide a scale-up evaluation with a high statistical power and a high temporal resolution, and they are also time-consuming and expensive. Continuous monitoring and quantification of protein biomarkers in these complex biofluids without perturbing the living cells remain challenging.

[0007] Cytokines are small (6-70 kDa) proteins that affect nearly every biological process, ranging from embryonic development to disease pathogenesis and aging. Cytokine-mediated intercellular communication is the main mechanism by which immune cells communicate with each other. Understanding the dynamics of cytokine secreted by immune cells over time is important for revealing the functionality of cytokine in an immune response. The most commonly used methods for cytokine quantification are the enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Various biosensors based on protein arrays and bead-based immunoassays have been reported to enable simultaneous quantification of multiple cytokines. The low detection limit for most high-sensitivity immunoassays and biosensors is typically in the range of 0.1-1 pg / mL. However, the dynamically secreted concentration of cytokines in biofluids is often below 0.1 pg / mL and, therefore, not quantifiable. Several recent amplification technologies enabled the detection in the low femtogram / mL range through plasmon-enhanced fluorescence, photonic-plasmonic coupling, electrochemiluminescence, or molecular on-bead signal amplification. Such low detection limits were achieved with the sample incubation of 1 hour or longer and sample processing with reagent diluents. The long sampling time increases the sensitivity but limits the temporal resolution in continuous monitoring. Therefore, multiplexed biosensors to quantify trace amounts of cytokines in unprocessed biofluids with a short sampling time needs to be developed.SUMMARY

[0008] This summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it to be used as an aid in limiting the scope of the claimed subject matter.

[0009] In some aspects, a biosensor for in situ continuous monitoring of molecular biomarkers includes a flexible organic or inorganic layer, and a biorecognition element microarray placed on the organic or inorganic layer. In some aspects, the flexible layer is placed on a disk.

[0010] In some aspects, the disk is made of a material including one or more of polydimethylsiloxane (PDMS), silicone, polyurethane, polymers, and hydrogels.

[0011] In some aspects, the organic or inorganic layer enables the immobilization of biorecognition elements, such as polystyrene (PS) film comprises functional groups from Gamma irradiation to increase biorecognition element binding.

[0012] In some aspects, the biorecognition elements are selected from the group consisting of antibody, aptamer, peptide, molecularly imprinted polymers, nucleic acid and combinations thereof.

[0013] In some aspects, diameters of the PS film and the disk are between 0.1-100 mm in diameter.

[0014] In some aspects, the disk has a thickness of less than 2000 pm.

[0015] In some aspects, the PS film has a thickness of less than 50 pm.

[0016] In some aspects, the disk and the PS film are optically transparent.

[0017] In some aspects, the capture antibody microarray comprises IL-6 capture antibodies.

[0018] In some aspects, the capture antibody microarray comprises TNF-a capture antibodies.

[0019] In some aspects, a method of making a floating biosensor includes placing a microarray of capture antibodies on a PS film, and placing the PS film on top of a disk.

[0020] In some aspects, diameters of the PS film and the disk are between 3-10 mm in diameter.

[0021] In some aspects, the capture antibodies comprise one or more of IL-6 and TNF-a.

[0022] In some aspects, the disk comprises PDMS.

[0023] In some aspects, the method includes applying a coating of bovine serum albumin to the floating biosensor.

[0024] In some aspects, the PS fdm is cut to shape via laser pulses.

[0025] In some aspects, the disk has a thickness of less than 2000 pm.

[0026] In some aspects, the PS film has a thickness of less than 50 pm.

[0027] In some aspects, the disk and the PS film are optically transparent.

[0028] In some aspects, the microarray of capture antibodies comprises IL-6 capture antibodies.

[0029] In some aspects, the microarray of capture antibodies comprises TNF-a capture antibodies.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] A more complete understanding of the subject matter of the present disclosure may be obtained by reference to the following Detailed Description when taken in conjunction with the accompanying Drawings wherein:

[0031] Figure 1 is a schematic illustration of fabrication and application of a floating sensor for continuous monitoring of multiplexed protein biomarkers.

[0032] Figure 2A is a microarray design with the IL-6 capture antibody microdots and positive control markers; Figure 2B is a graph of IL-6 dose-dependent fluorescence intensity' on the floating sensors following FLISA; and Figure 2C is a graph of IL-6 dose-dependent absorbance intensity following standard ELISA.

[0033] Figures 3A-3C are graphs of IL-6 dose-dependent fluorescence intensity on floating sensors following FLISA with Figure 3A) showing 2-hour and Figures 3B and 3C showing 15- min sampling times.

[0034] Figure 4A is a graph of fluorescence intensities from IL-6 capture antibody arrays after exposure to different concentrations of IL-6 and TNF-a. Figure 4B is a graph of fluorescence intensities from TNF-a capture antibody arrays after exposure to different concentrations of TNF-a and IL-6. Figure 4C is a graph of TNF-a calibration curve using standard ELISA. Figure 4D is a graph of linear regression plot of IL-6 concentration in cell culture medium samples using standard ELISA and floating multiplex sensor. Figure 4E is a graph of linear regression plot of TNF- a concentration in cell culture medium samples using standard ELISA and floating multiplex sensor. Figure 4F is a graph of fluorescence intensities resulting from IL-6 at different concentrations measured from the sensor with and without glycerol preservation 24 h after the sensor preparation.

[0035] Figure 5A is a schematic of macrophage differentiation. Figure 5B is a graph of cell viability of macrophages with and without exposure to floating sensors. Concentrations of IL- 6 and TNF a secreted by MO (Figure 5C) and Ml (Figure 5D) macrophages quantified by multiplexed floating sensors.

[0036] Figure 6 is a graph of zoomed-in IL-6 dose-dependent fluorescence intensity at a low concentration range.

[0037] Figure 7A is a graph of IL-6 dose-dependent fluorescence intensity following pFLISA collected from 3 microdots on one sensor, and Figure 7B is a graph of IL-6 dose-dependent fluorescence intensity following pFLISA collected from 9 microdots of 3 sensors (3 microdots in each sensor).

[0038] Figure 8 is a graph of percent mean relative error and coefficient of variation in back- calculated concentrations at the concentration range from 0.1 pg / mL to 100 pg / rnL.

[0039] Figure 9 is a graph of IL-6 dose-dependent fluorescence intensity on floating sensors following pFLISA with 30-min sampling time.

[0040] Figure 10 is agraph of TNF a dose-dependent fluorescence intensity on floating sensors with pristine and Gamma irradiated PS following FLISA.

[0041] Figure 11 is a graph of IL-6 dose-dependent fluorescence digital counting following pFLISA with 15-min sampling time.

[0042] Figure 12A is graph of fluorescence intensities from mouse IL-6 capture antibodies after exposure to different concentrations of mouse and human IL-6 and TNF-a, and Figure 12B is a graph of fluorescence intensities from mouse TNF-a capture antibodies after exposure to different concentrations of mouse and human TNF-a and IL-6.

[0043] Figure 13 is a graph of fluorescence intensities resulting from IL-6 at different concentrations measured from freshly prepared sensors and glycerol-coated sensors after storage at 4 °C for 1 day and 3 days.

[0044] Figure 14 TNF-a dose-dependent fluorescence intensity on floating sensors with pristine and Gamma irradiated PS following pFLISA.DETAILED DESCRIPTION

[0045] It is to be understood that the following disclosure provides many different embodiments, or examples, for implementing different features of various embodiments. Specific examples of components and arrangements are described below to simplify the disclosure. These are, of course, merely examples and are not intended to be limiting. The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. Reference will now be made to more specific embodiments of the present disclosure and data that provides support for such embodiments. However, it should be noted that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way. Reference will now be made to particular materials and methods utilized by various embodiments of the present disclosure. However, it should be noted that the materials and methods presented below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0046] Disclosed is a biosensor to enable in-situ continuous monitoring of immune biomarkers at attomolar concentrations with 15 -minute temporal resolution. A biosensors implemented with an antibody array provides multiplexed detection and quantification of cytokines. Digital technology may be used to further improve the low detection limit. The biosensor can float on the surface of culture media without perturbing the living cells and quantify the time-varying cytokine concentrations from the same cell populations. The easy-to-apply biosensor with attomolar sensitivity and multiplexing abilities enables an in-situ analysis of proteinbiomarkers in various biofluids and tissues to aid the diagnosis and treatment of diverse diseases.

[0047] A buoyant (i.e., floating) sensor to enable multiplexed detection and quantification of cytokines in culture media with the attomolar detection limit lower than previously reported values is disclosed herein. The floating sensor operates by resting on the surface of the culture media for a short duration of 15 minutes during sampling, thereby not perturbing the living cells in the culture. The sensors introduced at different times can quantify the temporal changes in the concentration of cytokines secreted by the same cell populations. For a proof-of-concept demonstration, human interleukin 6 (IL-6) and tumor necrosis factor-a (TNF-a) served as model inflammation biomarkers. The short sampling time allows for continuous monitoring of IL-6 and TNF-a in the culture media with a high temporal resolution. The low limits of detection for IL-6 and TNF-a were found to be 3.5 fg / mL (148 aM) and 21 fg / mL (808 aM), respectively, with multiplexed biosensors. Ultrabright fluorescent nanolabels that can be counted with a standard fluorescent microscope to further improve sensitivity were used. With digital counting, the low detection limit for IL-6 was further reduced to 14 ag / mL (0.6 aM), which is the lowest reported concentration achieved by nanobiosensors. These sensing capabilities reveal that the TNF-a secretion by classically activated macrophages is -1400 times faster than IL-6 in the first two hours after the activation, which cannot be quantified by conventional approaches.

[0048] In various aspects, the floating sensor comprises a poly dimethylsiloxane (PDMS) disk, a flexible polystyrene (PS) film, and a capture antibody microarray printed on the PS film (see Figure I). In some aspects, the disk may be omitted and the flexible film may be applied directly to, for example, a wound dressing. In some aspects, the disk may be made of one or more of PDMS, silicone, polyurethane, hydrogels, silicon, silica, and various polymers (e.g., polyester, polyethylene, terephthalate, polycarbonate, polyimide, polyethylene, polyvinyl chloride, polyacrylate, polypropylene). In some aspects, the biorecognition elements on the flexible film may be applied without printing (e.g., the biorecognition elements may be applied by immersion, dip coating, spray coating, drop casting, and the like). In other aspects, the film may comprise any of a variety of organic or inorganic materials. Organic materials include polystyrene, polyimide, polyethylene terephthalate, polyvinylidene fluoride, parylene, polyethylene naphthalate, poly(2-hydroxyethyl methacrylate), polyamide, polyester, polyvinylchloride, polycarbonate, poly methyl methacrylate, and combinations thereof.Inorganic materials include gold, silver, zinc oxide, aluminum oxide, silicon dioxide, silicon nitride, and combinations thereof. PDMS is biocompatible, hydrophobic, and has a low density (0.97 kg / m3), allowing the sensor to float on the surface of cell culture media without disturbing the living cells. In solid-phase immunoassay, polystyrene is a commonly used material that can be modified with various approaches to alter biomolecule binding affinity. Antibodies bind to pristine PS primarily through hydrophobic interactions. Previous studies showed that the Gamma irradiated PS surface provided higher binding affinity for antibodies and antigens, thereby improving immunoassay sensitivity'. The PS surface was treated with Gamma irradiation to increase capture antibody binding. Gamma irradiation oxidizes the PS surface and introduces functional groups, which probably include carbonyl, hydroxyl, and carboxyl groups. Following the Gamma irradiation, capture antibodies can bind to the PS surface with enhanced affinity through the combined hydrophobic and ionic interactions. To demonstrate the multiplexed capability, we chose human IL-6 and TNF-a as model inflammation biomarkers. Dynamic changes in the concentrations of these cytokines in various biofluids, including cell culture media, interstitial fluid, and blood, are associated with various biological and physiological conditions. The IL-6 and TNF-a secretions are extremely dynamic, and the half-lives of IL-6 and TNF-a are 15.5 h and 18.2 min, respectively. To detect and quantify these two cytokines simultaneously, we printed the IL-6 and TNF-a capture antibody microarrays on the PS surface in each sensor. Subsequently, the sensor was set afloat on the culture media surface for 15 min to capture IL-6 and TNF-a, followed by the plasmon- enhanced fluorescence-linked immunosorbent assay (pFLISA). The sensors introduced at different times quantified the temporal changes in the cytokine concentration from the same culture. After capturing the cytokine targets during floating, the sensors were incubated with IL-6 and TNF-a detection antibodies and then fluorescent nanolabels. These ultrabright fluorescent nanolabels, called plasmonic-fluors (PFs), are comprised of a plasmonic nanostructure coated with fluorescent dye IR650 and streptavidin (PF-650). Previous studies show that PFs are nearly 10,000-fold brighter compared to the corresponding conventional fluorophores.

[0049] In some aspects, the sensor includes a PS film laminated on a PDMS disk. The PDMS disk is, for example, about 5 mm in diameter and 600 pm in thickness, and the PS film has the same diameter and is about 30 pm thick, which can fit into a 96-well microplate. In various aspects, diameters of the PS film and disk may be between about 0. 1 mm to 100 mm. In various aspects, the disk has a thickness of less than 1000 pm, and / or the PS film has a thickness ofless than 100 pm. which helps with an overall flexibility of the biosensor. Both components are optically transparent and ideal to use in pFLISA. The PDMS disk was fabricated with die cutting of the PDMS sheet, which provides flexibility in tailoring its diameter and thickness for different applications. The PS circular fdms were prepared with picosecond-fast laser pulses to avoid plastic deformation and material degradation. It is important that the flexible PS fdm seamlessly adheres to the PDMS surface to eliminate undesired fluorescence backgrounds and ensure reliable sensor performance. The PS circular films prepared by a CO2 laser cutter exhibited material degradation and plastic deformation at the edge, resulting in a gap between the PS and the PDMS surface. Such a nonconformal interface results in unwanted nonspecific binding and fluorescence backgrounds. An improved cut can be achieved using a picosecondfast laser cutter (e.g., LPKF Protolaser R4). In some aspects, short pulses of the laser can be used to create a precise, clean cut compared to the CO2 laser cutter. The sensor can be easily picked up by a vacuum pick-up tool and placed on the surface of culture media without disturbing the living cells.

[0050] We designed and printed antibody microarrays to achieve multiplexed quantification of cytokine targets. Droplet jetting is a powerful approach to print and pattern different functional materials on various substrates, including glass slides, polystyrene, filter paper, and nitrocellulose membrane. Figure 2A shows 6 x 6 microdot array with asymmetrical spatial control markers to determine the location of each microdot for quantification. We printed IL-6 capture antibody microarray on the PS surface to characterize and optimize the sensor performance. Antibody droplets of 3 nL yield individual microdots with a diameter of -220 pm and a center-to-center distance of 500 pm. Biotinylated antibodies were printed on the control spots, which bind to fluorescent labels conjugated with streptavidin. After printing, the PS surface was blocked with BSA for 1 hour and then exposed to varying concentrations of IL-6 in reagent diluent (lwt% BSA in IX phosphate-buffered saline), followed by binding of biotinylated detection antibody to the captured IL-6 and fluorescent labeling. The PF nanolables were synthesized following the previous protocols (see details herein). Silver- coated gold nanorods (AuNRs@Ag) with a longitudinal plasmonic resonance wavelength of 650 nm were synthesized to overlap with the Fluorescent dye IR650 excitation and emission. Subsequently, the AuNR@Ag nanocuboids were coated with an ~3 nm thick siloxane copolymer layer and BSA-conjugated IR650 and streptavidin. Fluorescent dye Cy5 exhibits maximum absorption and emission at 649 nm and 666 nm. respectively. Subsequently, the siloxane surface was functionalized with BSA, Cy5, and streptavidin for further use.

[0051] Following pFLISA, we collected the fluorescence images of the microarray on the floating sensors after exposing them to changing concentrations of IL-6 from 1 ng / mL to 1 fg / mL for 2 hours. The blank control represents the biosensor exposed to the reagent diluent without IL-6. The microdots located at different regions show uniform fluorescent intensity, supporting the capability for spatially multiplexed quantification. The fluorescent intensity increases with the increased IL-6 concentration, while the control spots always show reliable high fluorescent intensity. The fluorescent intensity from microdots in the blank control is comparable to the background, confirming low nonspecific binding of the fluorescent labels. Fluorescence intensity averaged individual microdots (N=3) w as used for the quantification of IL-6 concentration (see Figure 2B). Calibration curves were fitted with a five-parameter- logistic method. The low limit of detection (LLOD, defined as mean + 3<r of the blank) and the low' limit of quantification (LLOQ, defined as mean + 1 OCT of the blank) were determined to be 0.51 and 49 fg / mL for IL-6, respectively. Magnified fluorescence images and calibration curve (see Figure 6) showed that the fluorescence intensity corresponding to 100 fg / mL of IL-6 is much higher than the blank. In addition, the LLOQ obtained from 3 microdots of one sensor is similar to that from 9 microdots of 3 sensors, indicating the high batch-to-batch consistency (see Figure 7). In addition to LLOD and LLOQ, we calculated the mean relative error to be less than ±8% and the coefficient of variation to be less than 24% at the concentration range from 0.1 pg / mL to 100 pg / mL, based on the validation method recommended previously (see Figure 8). For comparison, the ELISA provides the LLOD and LLOQ of 0.71 and 2.2 pg / mL, 1392- and 45-fold higher than those of pFLISA (Figure 2C). These results confirm the higher sensitivity of buoyant sensors based on pFLISA compared to ELISA.

[0052] In continuous monitoring, temporal resolution is important to reveal dynamic changes in the concentration of cytokines in biofluids. Standard ELISA typically involves 2 hours of sample incubation to achieve the desired sensitivity. Here, we examined the effect of sampling time on sensitivity by reducing the sensor floating duration from 2 hours to 15 minutes. In these experiments, the sensors floated on the surface of culture media spiked with varying concentrations of IL-6 and were kept at 37°C in an incubator, consistent with cell culture experimental conditions. Figure 3 A shows a calibration curve of averaged fluorescent intensity as a function of IL-6 concentrations from 10 ng / mL to 1 fg / mL w ith a 2-hour sampling time. The LLOD and LLOQ were calculated to be 1.4 fg / mL and 29 fg / mL. As expected, shorter sampling times decreased the fluorescence intensity and increased the LLOD and LLOQ following the reduced number of captured IL-6 targets (see Figure 3B and Figure 6). With 15-min sampling time, the LLOD and LLOQ increased to 3.0 fg / mL and 69 fg / mL, respectively, twice as high as those with 2-hour sampling but still much lower than those provided by ELISA with 2-hour sampling.

[0053] Immunoassays based on digital imaging technologies can provide higher sensitivity than ensemble average measurements by counting the labels linked to the binding of biomolecule analytes. To further decrease the LLOD, we performed digital counting of PF nanolabels from fluorescence microscope images collected at the center of the microdots corresponding to the 15 min sampling (see Figure 3D). Fluorescence images were taken with a field of view 375 / zm x 375 / m corresponding to IL-6 concentrations from 10 pg / mL to 1 fg / mL. With decreasing IL-6 concentration, decreased numbers of PF nanolabels were observed. After processing the images by first Gaussian blur, where the high-frequency noise was filtered, the bright individual dots could be visualized and counted. This method was sensitive at low concentrations while saturated at concentrations of >100 pg / mL (see Figure 10). A calibration curve was fitted using a linear function, and the LLOD and LLOQ were calculated to be 0.014 and 0.031 fg / mL, respectively (see Figure 3C). The fitting quality can be improved after removing the blank data point from the calibration curve, although this slightly increased the LLOD and LLOQ to 0.096 and 0. 18 fg / mL (see Figure 11). This confirms that the digital counting approach can further decrease the LLOD and LLOQ and expand the dynamic range by ~3 orders of magnitude for the quantification of cytokines based on pFLISA.

[0054] Next, we demonstrate the spatial multiplexing capability of the buoyant sensors for the simultaneous quantification of IL-6 and TNF-a. To examine the possible cross-reactivity, we exposed the multiplexed sensor to a series of varying concentrations ofIL-6 and TNF-a spiked in culture media and quantified the fluorescence intensity following pFLISA. Figure 4 A shows the fluorescence intensity of IL-6 antibody microdots exposed to varying concentrations of IL- 6 and TNF-a. The fluorescence intensity resulting from the binding of TNF-a at all concentrations is low. The fluorescence intensity corresponding to increasing concentration of IL-6 shows a consistent trend with singleplex sensors and only a slight increase in the LLOQ from 69 to 140 fg / mL. Similarly, the fluorescence intensity of TNF-a antibody microdots was quantified following the exposure of varying concentrations of IL-6 and TNF-a (see Figure 4B). The LLOD and LLOQ for TNF-a were calculated to be 21 fg / mL and 0.37 pg / mL, respectively. In comparison, the standard ELISA provides the higher LLOD and LLOQ of 0.33 and 1.1 pg / mL (see Figure 4C). The fluorescent intensity resulting from the binding of IL-6 tothe TNF-a antibody microdots is comparable to that from the binding of TNF-a at a low concentration of 0.1 pg / mL. We further validated the multiplexing capability with four cytokine targets, including human IL-6, human TNF-a, mouse IL-6, and mouse TNF-a, to challenge the cross-reactivity between different species. Our results show that mouse IL-6 and TNF-a antibodies do not exhibit cross-reactivity with human IL-6 and TNF-a, consistent with the statement from the vendor (see Figure 12). The LLOQ for mouse IL-6 and TNF-a was calculated to be 29 fg / mL and 0.49 pg / mL, respectively.

[0055] For performance validation, we simultaneously quantified human IL-6 and TNF-a in cell culture media collected from macrophage cell cultures using multiplexed buoyant sensors and standard ELISA. Linear regression between the ELISA and multiplex sensor quantifiedIL-6 and TNF-a concentrations shows high correlations with Pearson’s r values of 0.98 and 0.95, respectively (see Figures 4D and 4E). We assessed and validated the sensor accuracy through spike-and-recovery and linearity-of dilution experiments and found recovery rates in the 85% to 105% range (see Tables 1 and 2 below).

[0056] Table 1: Spike and recovery of IL-6 in macrophage culture supernatant.

[0057] Table 2: Linearity-of-dilution results for IL-6 in macrophage culture supernatant.

[0058] These results confirm the effectiveness of using the pristine culture medium for calibration to quantify the analyte in the macrophage culture supernatant. To improve thestability of printed antibodies, a thin layer of glycerol was spin-coated on the PS surface after printing. The sensors show only a slight degradation in sensitivity after storage at room temperature for 24 h compared with the freshly prepared sensors. Without this preservation strategy, the sensors exhibited significant degradation in sensitivity and a detectable fluorescence signal was observed only at high concentrations of IL-6 (>10 pg / mL) due to the denaturation of the immobilized capture antibody (Figure 4F). Storing the glycerol-coated sensors at 4 °C improves the antibody stability. The sensors showed similar sensitivity after 1 day of storage at 4 °C and increased LLOQ from 29 to 95 fg / mL after 3 days (see Figure 13). Alternatively, other preservation strategies, such as molecular encapsulation and metal organic framework coating, can also be applied to further improve antibody stability.

[0059] As a proof of concept, we demonstrated the use of our buoyant sensors for in situ monitoring of IL-6 and TNF-a in macrophage cell cultures. We utilized a human leukemia monocytic cell line (THP-1) in these experiments, which is a well-established in vitro cell model to study monocyte and macrophage functions. Phorbol 12-myristate 13-acetate (PMA, 50 ng / mL) was used to differentiate the human THP-1 monocytes into macrophages (MO; Figure 5A). The second cell group is Ml macrophages classically activated with lipopolysaccharide (LPS) and interferon gamma (IFN-y). The sensor components were sterilized with 75% ethanol to avoid cell culture contamination. We evaluated the sensor biocompatibility by live / dead cell assay after floating the sensors on the surface of MO and Ml cell cultures for 7 h. Fluorescence images of cells after live / dead staining were taken. The cell morphology and dimensions in the standard cultures are consistent with those with sensors. The cell viability of both MO and Ml in all cases is higher than 96% (see Figure 5B). The sensor biocompatibility was further confirmed by the similar cell viability after 3 days of sensor incubation in cell culture as the control.

[0060] Macrophage Ml polarization leads to increased concentrations of proin fl ammatory cytokines, including IL-6 and TNF-a. However, the temporal concentration changes could only be measured every several hours from different cell populations with replicated cell culture conditions because of limitations with the existing bioanalytical approaches, including ELISA. In contrast, our approach can simultaneously quantify the secreted cytokine concentrations with a temporal resolution of 1 h or a shorter time interval from the same cell populations. After immobilizing M0 for 24 h, the cell culture media with PMA were changed with new media with and without LPS and IFN-y. Subsequently, 8 buoyant sensors were introduced onthe culture media surface every hour and incubated for 15 min, and the dynamic changes in the IL-6 and TNF-a levels were monitored over ~7 h. In the MO macrophage cell culture, the IL- 6 concentration stayed below 0. 1 pg / mL and slightly decreased below 10 fg / mL after 5 h, while the TNF-a concentration gradually increased by 2 orders of magnitude from 0. 12 pg / mL to 12 pg / mL in 7 h (see Figure 5C). The results indicate a low level of pro-inflammatory cytokine secretion in MO macrophages. In contrast, in the presence of LPS and IFN-y, macrophages secreted high levels of proinflammatory cytokines (see Figure 5D). The IL-6 concentration increased by more than 4 orders of magnitude from 39 fg / mL to 590 pg / mL in 6 h. The TNF- a concentration increased from 74 fg / mL to 1.3 ng / mL in 3 h and reached a plateau. It is important to note that the initial low concentrations of IL-6 and TNF-a are not detectable and quantifiable with ELISA.

[0061] The introduction of a nonperturbing biosensor enables the quantification of protein analytes at attomolar concentrations with high temporal resolution. The biosensor can interface with the biofluids and capture target biomarkers in situ without disrupting biological systems. The biosensor with antibody arrays enables the simultaneous quantification of multiple cytokines. With digital counting, the LLOD for IL-6 was below 1 aM and 246-fold lower than the LLOD defined by the average fluorescence intensity analysis. The record high sensitivity reported here mainly results from the quantification approach via digital counting ultrabright nanolabels, high binding properties of the Gamma irradiated PS surface, and strong affinity between the antibody and target proteins. Such a strong affinity enables the capture of target proteins at veiy low concentrations while making their continuous monitoring challenging without a refreshing mechanism. Our ongoing efforts focus on refreshing the binding sites to release captured molecules for continuous monitoring. With its attomolar sensitivity and multiplexing capabilities, this user-friendly biosensor offers a valuable tool for in situ analysis of protein analytes in various biofluids and tissues.

[0062] Experimental section

[0063] Materials. Microplates, capture antibodies, biotinylated detection antibodies, IL-6, TNF-a, horseradish peroxidase (HRP)-labeled streptavidin, color reagents, and stop solution (2N H2SO4) were purchased from R&D Systems. Phosphate-buffered saline (PBS, 10X) was purchased from Invitrogen. BSA was purchased from Sigma-Aldrich. Tween-20 was purchased from Fisher Scientific. Dow SYLGARD™ 184 Silicone was purchased from Ellsworth Adhesives. Polystyrene film was purchased from Goodfellow. THP-1 cells andRoswell Park Memorial Institute (RPMI)-1640 media were purchased from ATCC. The fetal bovine serum (FBS) was purchased from R&D. Penicillin / streptomycin (pen-strep) was purchased from Thermo Fisher. PMA and LPS were purchased from Sigma Aldrich. IFN-y was purchased from Cell Signaling Technology. Plasmonic-fluor nanolabels (PF650TM ultrabright fluor), synthesized according to our previous report, were obtained from Auragent Biosceince LLC (St. Louis, USA).

[0064] Sensor fabrication: For PDMS preparation, SYLGARD™ 184 Silicone Elastomer base and curing agent were mixed at a 10:1 ratio, degassed, transferred into Petri dishes to form a 600-pm thick sheet, and then kept for curing at 60°C for 12 hours. Next, the PDMS sheet was cut into circular disks of 5 mm in diameter with a metal disc cutter. The PS was treated with Gamma irradiation and cut with picosecond-fast laser pulses (frequency: 300 kHz; power: 2 W; mark speed: 400 mm / s; repetitions: 4). The sensors prepared with pristine PS were found to have lower sensitivity than those prepared with Gamma-irradiated PS (See Figure 14). The PDMS and PS were sterilized with 75% ethanol and then assembled using a vacuum pick-up tool to form buoyant sensors. Capture antibodies (120 pg / mL in IX PBS for IL-6 and 240 pg / mL in IX PBS for TNF-a) were printed on the PS surface using an electromagnetic droplet jetting nozzle and incubated for 30 mins at room temperature. Next, the buoyant sensors were rinsed with washing buffer (0.05% Tween-20 in IX PBS) 3 times and blocked with 3 wt% BSA in lx PBS for one hour.

[0065] Human IL-6 and TNF-a ELISA. The ELISA procedure follows the optimized protocol provided by the vendor (R&D Systems). A 96-well microplate was first coated with capture antibody (2 pg / mL in IX PBS for IL-6 and 4 pg / mL in IX PBS for TNF-a) through overnight incubation. Next, the plate was rinsed with washing buffer (0.05% Tween-20 in IX PBS) 3 times and blocked with 3 wt% BSA in IX PBS for one hour. After rinsing, varying concentrations of IL-6 and TNF-a spiked in reagent diluent (lwt% BSA in IX PBS) or cell culture medium (RPMI 1640 medium supplemented with 10% FBS and 1% pen-strep), and the samples collected from cell cultures were added to the microplate wells. After 2 hours of incubation, the microplate was rinsed and incubated with the biotinylated detection antibody (50 ng / mL in reagent diluent for both IL-6 and TNF-a) for 2 hours. Subsequently, the microplate was rinsed and incubated with HRP-labelled streptavidin for 20 minutes. After rinsing, the substrate solution (1 : 1 mixture of H2O2 and tetramethylbenzidine) was added to the microplate to allow an enzymatic reaction followed by a stop solution after 20 minutes. All theincubation steps were at room temperature without disturbance. The optical density was immediately determined using a microplate reader (Tecan Infinity M Nano Plus).

[0066] Human IL-6 and TNF-a pFLISA: The sensors were exposed to 100 pL varying concentrations of IL-6 and TNF-a spiked in reagent diluent (lwt% BSA in IX phosphate- buffered saline) or cell culture media in a floating format with a sampling duration from 15 minutes to 2 hours at 37°C. Subsequently, the buoyant sensors were transferred into a 96-well microplate and rinsed with wash buffer (0.05% Tween-20 in IX PBS) 3 times. The sensors were then incubated with the biotinylated detection antibody (50 ng / mL in reagent diluent for both IL-6 and TNF-a) for 2 hours, followed by the wash buffer rinsing 3 times, 100 / / L PF nanolabel solution (optical density ~0.5) was added and incubated for 30 minutes, followed by rinsing 3 times. Finally, the sensors were imaged using LI-COR fluorescence imager and BioTek Cytation 5 Cell Imaging Multi-Mode Reader. For imaging analysis, at least three microdots were used to obtain mean fluorescent intensity and nanolabel numbers and corresponding standard deviations. To monitor IL-6 and TNF-a in macrophage culture, the sensors were introduced to the surface of culture media at different times and floated for 15 minutes to capture the targets. All other steps follow the same for IL- 6 and TNF-a quantification. To validate the sensor accuracy, we performed spike-and recovery and linearity- of-dilution experiments. In spike-and-recovery experiments, 90 LIL macrophage culture supernatant was spiked with 10 pL pristine cell culture medium (RPMI- 1640 with 10% FBS and 1% pen-strep) containing 0, 250 pg / mL, 500 pg / mL, and 1 ng / mL IL- 6. Standard IL-6 in pristine culture medium at concentrations of 0, 25 pg / mL, 50 pg / mL, and 100 pg / mL served as controls. The IL-6 concentrations in the controls and samples were quantified using our sensors, and percentage recovery was calculated using the equation: Recovery rate = [(observed sample spike concentration - observed sample initial concentration) / expected concentration] x 100% (see Table 1). In linearity-of-dilution experiments, macrophage culture supernatant was diluted with pristine culture medium at different dilution factors of 1:2, 1:4, and 1:8 and then quantified with our sensors. Percentage recovery was calculated using the equation: Recovery rate = (observed sample concentration / expected concentration) x 100% (see Table 2).

[0067] Macrophage culture: The THP-1 cell culture follows the previous protocols. RP MI- 1640 media supplemented with 10% FBS and 1% pen-strep was used to culture THP-1 cells. For differentiation into macrophages, the THP-1 cells cultured in the 24-well microplate witha cell density of 200,000 cells / cm2were treated with the culture medium containing 50 ng / mL PMA. It took 24 hours for the THP-1 cells to differentiate and adhere to the microplate. The fresh cell culture media (RPMI 1640 medium supplemented with 10% FBS and 1% pen-strep) with 100 ng / mL LPS and 15 ng / mL IFN-y were added to classically activate M0 into Ml. For live / dead viability staining, the cells were rinsed twice with sterile Dulbecco's PBS (DPBS) to thoroughly remove the culture medium and then immersed in the mixture of Calcein AM (2 / ;M in DPBS) and Ethidium Homodimer- 1 (4 / zM in DPBS) for 30 minutes at room temperature. The cells were imaged under a fluorescence microscope (Zeiss Axio Observer 3) with red (ex / em 495 nm / 635 nm) and green (ex / em 495 nm / 515 nm) channels to observe dead and live cells, respectively.

[0068] Although various embodiments of the present disclosure have been illustrated in the accompanying Drawings and described in the foregoing Detailed Description, it will be understood that the present disclosure is not limited to the embodiments disclosed herein, but is capable of numerous rearrangements, modifications, and substitutions without departing from the spirit of the disclosure as set forth herein.

[0069] The term “substantially” is defined as largely but not necessarily wholly what is specified, as understood by a person of ordinary skill in the art. In any disclosed embodiment, the terms “substantially”, “approximately”, “generally”, and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0070] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the disclosure. Those skilled in the art should appreciate that they may readily use the disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the disclosure. The scope of the invention should be determined only by the language of the claims that follow. The term “comprising” within the claims is intended to mean “including at least” such that the recited listing of elements in a claim are an open group. The terms “a”, “an”, and other singular terms are intended to include the plural forms thereof unless specifically excluded.

[0071] Conditional language used herein, such as, among others, “can”, “might”, “may”, “e.g.”, and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or states. Thus, such conditional language is not generally intended to imply that features, elements and / or states are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or states are included or are to be performed in any particular embodiment.

[0072] While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the devices or algorithms illustrated can be made without departing from the spirit of the disclosure. As will be recognized, the processes described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used or practiced separately from others. The scope of protection is defined by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0073] Although various embodiments of the method and apparatus of the present invention have been illustrated in the accompanying Drawings and described in the foregoing Detailed Description, it will be understood that the invention is not limited to the embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the spirit of the invention as set forth herein.

Claims

CLAIMSWhat is claimed is:

1. A biosensor for in situ continuous monitoring of molecular biomarkers, the biosensor comprising: a flexible organic or inorganic layer; and a biorecognition element coating or microarray positioned on the organic or inorganic layer.

2. The biosensor of claim 1, further comprising a disk made of one or more of polydimethylsiloxane (PDMS), silicon, silica polyurethane, hydrogels, polyester, polyethylene, terephthalate, polycarbonate, polyimide, polyethylene, polyvinyl chloride, polyacrylate, and polypropylene.

3. The biosensor of claim 1, wherein the organic or inorganic layer enables the immobilization of biorecognition elements, such as polystyrene (PS) film and comprises functional groups from Gamma irradiation to increase biorecognition element binding.

4. The biosensor of claim 1, wherein the biorecognition elements comprise one or more of antibodies, aptamers, peptides, molecularly imprinted polymers, nucleic acids, proteins, enzymes and haptens.

5. The biosensor of claim 3, wherein the PS film comprises a diameter between 0. 1mm to 100 mm in diameter.

6. The biosensor of claim 2, wherein the disk has a thickness of less than 1000 pm.

7. The biosensor of claim 3, wherein the PS film has a thickness of less than 50 pm.

8. The floating biosensor of claim 3. further comprising a disk upon which the flexible organic or inorganic layer is placed, and wherein the disk and the PS film are optically transparent.

9. The biosensor of claim 1, wherein the capture antibody microarray comprises IL-6 capture antibodies.

10. The biosensor of claim 1, wherein the capture antibody microarray comprises TNF-a capture antibodies.

11. A method of making a floating biosensor, the method comprising: placing a microarray of capture antibodies on a PS film; and placing the PS film on top of a disk.

12. The method of claim 11. wherein the capture antibodies comprise one or more of IL-6 and TNF-a.

13. The method of claim 1 1 , wherein the disk comprises PDMS.

14. The method of claim 11, further comprising applying a coating of bovine serum albumin to the floating biosensor.

15. The method of claim 11, wherein the PS film is cut to shape via laser pulses.

16. The method of claim 11, wherein the disk has a thickness of less than 1000 pm.

17. The method of claim 11, wherein the PS film has a thickness of less than 100 pm.

18. The method of claim 11, wherein the disk and the PS film are optically transparent.

19. The method of claim 11. wherein the microarray of capture antibodies comprises IL-6 capture antibodies.

20. The method of claim 11, wherein the microarray of capture antibodies comprises TNF- a capture antibodies.

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