Microneedle patch for the ultrasensitive quantification of protein biomarkers in interstitial fluid

Abstract

The detection and quantification of protein biomarkers in interstitial fluid (ISF) is hampered by challenges in its sampling and analysis. Here, we report a microneedle patch for the fast in vivo sampling and on-needle quantification of target protein biomarkers in ISF. We used plasmonic fluor — an ultrabright fluorescent label — to improve the limit of detection of various ISF protein biomarkers by nearly 800-fold with respect to conventional fluorophores, and a magnetic backing layer to implement conventional immunoassay procedures on the patch and thus improve measurement consistency. We used the microneedle patch in mice for the minimally invasive evaluation of the efficiency of a cocaine vaccine, for the longitudinal monitoring of the levels of inflammatory biomarkers, and for the efficient sampling of the calvarial periosteum — a challenging site for biomarker detection — and quantification of its levels of the matricellular protein periostin, which cannot be accurately inferred from blood or other systemic biofluids. Microneedle patches for the minimally invasive collection and analysis of biomarkers in ISF might facilitate point-of-care diagnostics and longitudinal monitoring.

Interstitial fluid (ISF), among various peripheral biofluids such as saliva, sweat, and tears, is a particularly rich source of soluble bioanalytes including proteins, peptides, metabolites and nucleic acids, which exhibits close correlation with blood.^1–6 It also represents the locoregional biomolecular composition of specific tissues of interest, such as within the tumor microenvironment.⁷ Simple and effective methods that enable comprehensive analysis of ISF can lead to transformative advances in novel biodiagnostic technologies that are not only minimally-invasive and pain-free, but also ideally suited for point-of-care (POC) and resource-limited settings.^8–10 Extraction of ISF followed by ex vivo analysis¹¹ has not been widely embraced in both pre-clinical and clinical applications due largely to (i) difficulty in extracting ISF, which is time-consuming and requires bulky instruments^12–14; and (ii) extremely small amount of ISF that can be extracted using current technology, making comprehensive analysis challenging¹⁵. For example, microneedle-assisted extraction of ISF yields only about 2 μL of biofluid from 4 cm² of human skin even after 20 minutes of vacuum suction, which is simply insufficient for comprehensive proteomic and metabolomic analysis.¹⁵ In fact, in pre-clinical settings (e.g., small animal models), to measure the concentration of target biomarkers, pooling adequate amount of ISF from multiple subjects is common, which inevitably masks the subject-to-subject biological variability.¹⁶

In contrast to ISF extraction, microneedles functionalized with biorecognition elements can specifically capture target biomarkers in ISF, followed by ex vivo analysis.^17–18 Direct exposure of microneedles to ISF allows the biorecognition elements on the microneedle to capture target biomarkers in situ, thus offering a promising technology for simple and efficient biodetection. However, physiological concentrations of the protein biomarkers in the ISF is usually lower when compared to that in blood.^{4, 19} Moreover, analyte-antibody binding kinetics are significantly deteriorated due to the “dense” tissue environment, which results in slower diffusion of target biomolecules to the sensor surface (i.e. microneedle surface), further lowering the probability of analyte capture and consequent signal intensity corresponding to the analyte. These challenges exacerbate the difficulty in detection of protein biomarkers in interstitial fluid. Despite the recent advances in multiplexed detection of biomarkers,²⁰ the sensitivity of existing microneedle-based analytical methods are insufficient to detect (or quantify) most ISF protein biomarkers, which limits the development potential diagnostic tests based on ISF biomarker levels. Most previous reports are limited to mice that have been intravenously injected with high concentrations of recombinant target markers as pseudo models, or to biomolecules present at relatively high levels (μg/ml in blood).¹⁷ Lastly, existing microneedle-based in vivo sampling and detection methods are limited to qualitative analysis in which the target biomarker concentration is represented as relative fluorescence intensity, absorbance value or normalized relative quantity.^{18, 20–21} This limitation precludes quantitative comparisons of the biomarker concentrations across different experiments and across different labs in biomedical research and decreases opportunities for standardization of the cut-off values for clinical biomarkers.

Here, we demonstrate ultrasensitive and quantitative measurement of target protein biomarkers in ISF through microneedle-based in vivo sampling and subsequent on-needle analysis. To improve the sensitivity of our microneedle-based immunoassay, we utilized an ultrabright fluorescent nanolabel, termed plasmonic-fluor, which improved the limit-of-detection of various ISF protein biomarkers by nearly 800-fold compared to conventional fluorophores and significantly shortened the sampling. Moreover, by harnessing the bilayer design of the microneedle, we were able to replicate conventional immunoassay procedures on microneedle patches, including a calibration curve based on “standard micropatches”. Using a series of mouse models, we demonstrate that the microneedle patch can be used for ultrasensitive and quantitative monitoring of various protein biomarkers through a simple stick-and-peel process. First, we probed the efficiency of a cocaine vaccine by monitoring cocaine-specific antibodies in dermal ISF. Second, we demonstrate sensitive detection and longitudinal monitoring of inflammatory biomarker levels in mice after induction of endotoxin-mediated shock. Last, we validated the application of the microneedle patch in the detection and quantification of the matricellular protein periostin in the calvarial periosteum, a novel but challenging detection site, using both control wild type (WT) and periostin knock-out (Postn^KO) mice. The minimally invasive microneedle patch obviates the need for destruction of target tissues and repeated blood-drawing in a short period, which can cause poor patient compliance or potential death of experimental mice in preclinical settings. This ultrasensitive biodetection technology can broadly enable the use of microneedle patches for biomarker collection and analysis.

Results

Design and fabrication of the microneedle patch

The novel biodetection technology introduced in this study relies on microneedles functionalized with biorecognition elements (e.g. antibodies) that penetrate the stratum corneum (or periosteum) and selectively capture protein biomarkers in the local ISF in a concentration-dependent manner. Subsequently, the microneedle patch was peeled off from the skin and the protein biomarkers bound on the microneedles were quantified by an ultrasensitive fluoroimmunoassay implemented ex vivo (Fig.1a). For efficient capture of target biomarkers in vivo, microneedles are required to exhibit high protein/antibody binding ability, high mechanical strength, and biocompatibility. Owing to its low cost, facile processability, and hydrophobic nature, polystyrene is widely utilized for microtiter plates in biomedical research and clinical diagnostics. Affinity reagents such as capture antibodies and blocking proteins can be efficiently immobilized on the polystyrene surface owing to the hydrophobic interactions between polystyrene and non-polar residues of the proteins. Thus, we have employed polystyrene for the fabrication of the microneedles, which were subsequently coated with capture antibodies to enable specific binding of the target biomarkers.

Fig. 1 |. Bilayered microneedle fabrication and material characterization.

a, Schematic illustration showing work flow of microneedle-based biodetection involving in situ sampling and on-needle detection of protein biomarkers in intersititial fluid (ISF). b, Schematic illustration of the fabrication steps of the bilayered microneedle patch. c, Optical image of a microneedle patch with the magnetic backing layer (black) and pristine polystyrene needles (hazy). Scale bar, 500 μm. d, Schematic illustration depicting the importance of employing microneedle patch with embedded magnetic nanoparticles, which facilitates various standard immunoassay procedures such as incubation and washing steps, and overcomes low-efficient patch-by-patch handling. e, SEM image of as-fabricated microneedle patch. Scale bar: 100 μm. Inset image shows side veiw of microneedle. Scale bar: 200 μm. f, Fluorescence microscopy images demonstrating efficient and uniform adsorption of antibodies on polystyrene microneedles. Scale bar, 500 μm. Inset image shows side veiw of fluorescence signal on microneedle. Scale bar: 200 μm.

The microneedle patch was fabricated using a silicone mold via two successive drop-casting steps (Fig.1b). Polystyrene solution (25% w/v in dichloromethane) was first cast on a silicone mold and the solvent was allowed to evaporate slowly under ambient conditions. Subsequently, a backing layer comprised of the mixture of polystyrene and magnetic nanoparticles (Fe₃O₄ nanoparticles) was formed on top of the pristine polystyrene layer. Incorporation of the magnetic nanoparticles in the backing layer is important to ensure that the microneedle patches stay at the bottom of a microtiter plate in the presence of a magnet underneath during subsequent immunoassay procedures (Fig.1b, c, and Supplementary Fig.1). This bilayer design overcomes the low throughput and poor reproducibility of patch-by-patch handling (i.e. incubation and wash steps performed manually one patch at a time), making the microneedle-based assay highly consistent, fast and reproducible (Fig. 1d and Supplementary Fig.2). The patch is comprised of an array of microneedles with a center-to-center distance of 600 μm (Fig.1e). Each microneedle is conical in shape with a 4 μm radius of curvature at the tip, a diameter of 300 μm at the base, and around 600 μm in height (Fig.1e, and Supplementary Fig.3). To assess the density and uniformity of antibody coating on polystyrene microneedles, we coated the microneedles with biotinylated anti-mouse IgG followed by blocking with bovine serum albumin (BSA). Subsequently, the microneedle patches were exposed to dye (LT680)-labeled streptavidin, resulting in a strong and uniform fluorescence signal along the entire length of the microneedle suggesting the uniform coating of the antibodies and BSA (as blocking layer) on the polystyrene microneedle surface (Fig.1f). In contrast, microneedles coated with BSA which subsequently exposed to LT680-streptavidin exhibited extremely weak fluorescence intensity (155-fold lower, signal close to pristine microneedle) (Supplementary Fig.4), suggesting low auto-fluorescence of the BSA and polystyrene microneedle surface.

Plasmonic-fluor linked immunosorbent assay (p-FLISA) on the microneedle patch

Conventional sandwich enzyme linked immunosorbent assay (ELISA) involves an enzymatic reaction that results in the formation of a soluble colored product in an analyte concentration-dependent manner. While highly standardized and routinely implemented in microtiter plates comprised of identical sampling wells, this approach is unsuitable for the microneedle patches due to (i) the relatively low sensitivity stemming from the limited sampling surface area (analyte present only on the micro-sized needles in real sampling situation), making the quantification of low-abundant analytes challenging; and (ii) the soluble nature of the colored product, which masks spatial variations in the amount of analyte bound across the patch, eliminating the possible spatial multiplexing capability. Hence, existing approaches are limited to pseudo mouse models involving high amounts of target analytes, which do not represent their true pathological and physiological concentrations. To overcome these challenges, we have employed novel fluorophore-linked immunosorbent assay (FLISA) that relies on “plasmonic-fluor” as an ultrabright and highly specific fluorescent nanolabel. Plasmonic-fluor is comprised of a gold nanorod coated with fluorophores (800CW) and a universal biological recognition element (e.g. biotin).²² BSA is employed as a scaffold to assemble all of these functional elements as well as to resist non-specific binding (Fig.2a, Supplementary Fig.5, see supplementary information for detailed description of plasmonic-fluor). Siloxane copolymer is employed as a spacer layer between the gold nanorod and the fluorophores to avoid metal-induced fluorescence quenching.²³ TEM images of the plasmonic-fluor confirmed the presence of a thin organic layer around the AuNRs (polymer and BSA conjugate) with an overall thickness of ~6.3±1.1 nm (Fig.2b, Supplementary Fig.6). Binding of plasmonic-fluor-800CW to strepdavidin-800CW coated at the bottom of a microtiter well resulted in a 1424-fold enhancement in the ensemble fluorescence intensity (Fig.2c, Supplementary Fig.7).

Fig. 2 |. Plasmonic-fluor enhanced fluorophore-linked immunosorbent assay (p-FLISA) on microneedle for ultrasensitive detection.

a, Schematic illustration of plasmonic-fluor as ultrabright fluorescence nanolabel. Plasmonic-fluor is comprised of a plasmonic core (gold nanorod (AuNR)), a polymer spacer layer, fluorophores, and a universal biorecognition element (biotin), which are assembled using BSA. b, TEM image of plasmonic-fluors. c, Fluorescence images and corresponding intensity of 800CW-streptavidin followed by the specific binding of plasmonic-fluor through biotin-streptavidin interaction, showing 1424-fold increase in fluorescence intensity. Error bars represent standard deviation. Data statistically significant P value = 0.0004, *** P< 0.001 by one-tailed unpaired t-test with Welch’s correction. Fluorescence intensity maps of mouse IL-6 (d) fluorophore-linked immunosorbent assay (FLISA) and (e) p-FLISA at various analyte concentrations. Scale bar: 500 μm. f, SEM images showing pristine microneedle (top) and after being probed by plasmonic-fluor on microneedle (bottom). Plasmonic-fluors have been highlighted by yellow arrows. Scale bar, 500 nm. Plots showing the IL-6 dose-dependent fluorescence intensity on microneedle from (g) conventional FLISA and (h) p-FLISA. Compared to FLISA, p-FLISA exhibited 790-fold lower LOD. i, Plot showing IL-6 dose-dependent optical intensity of ELISA implemented on microneedle. Error bars represent standard deviation. N=3 repeated tests.

To test the applicability of plasmonic-fluor as an ultrabright biolabel for use on a microneedle patch, we have employed mouse interleukin 6 (IL-6), a pro-inflammatory cytokine, as a representative protein biomarker. Conventional FLISA involves a standard sandwich immunoassay format of immobilizing the capture antibody on the surface of the microneedle, recognition and capture of analyte (IL-6), binding of biotinylated detection antibody to the captured analyte, exposure to streptavidin-fluorophore (800CW in this study), which binds to the biotin on the detection antibody with very high affinity. In contrast to conventional FLISA, plasmonic-fluor linked immunosorbent assay (p-FLISA) involves the use of plasmonic-fluor instead of a conventional fluorophore as the fluorescent label. To determine the improvement in sensitivity and limit-of-detection (LOD, defined as mean+3σ of the blank) of p-FLISA compared to FLISA, serial dilutions of IL-6 of known concentration (2.5 ng/ml to 2.5 pg/ml) were employed as standards. Fluorescence signals obtained after applying the plasmonic-fluor-800CW revealed nearly 530-fold enhancement in the ensemble fluorescence intensity compared to the conventional FLISA at the highest IL-6 concentration tested (2.5 ng/ml) (Fig.2d,e). Remarkably, the LOD of the IL-6 p-FLISA was found to be around 0.33 pg/ml, which is 790-fold lower compared to conventional FLISA (261 pg/ml) (Fig.2g, h). Scanning electron microscopy (SEM) images revealed the presence of plasmonic-fluors on the surface of the microneedle (Fig.2f, a few of plasmonic-fluors highlighted by yellow arrows). On the other hand, ELISA performed on the microneedle exhibited weak colorimetric signal and large standard deviation (Fig.2i). The optical density corresponding to the highest IL-6 concentration of ELISA on microneedle patch is nearly 10-fold lower than the standard ELISA implemented on a microtiter plate, approaching the background noise level (Fig.2, Supplementary Fig.8). The LOD of ELISA on the microneedle patch was measured to be around 53 pg/ml, which is 160-fold higher than that of p-FLISA (0.33 pg/ml) (Fig.2h, i).

Biophysicochemical properties of microneedle patches

Before deploying the microneedle patches for in vivo transdermal biodetection, we investigated their biophysicochemical properties such as (i) mechanical strength for penetration of dermal tissue; (ii) biocompatibility; and (iii) biosafety and potential side effects. To determine if the polystyrene microneedles possess sufficient mechanical strength to penetrate the skin under compression, we performed a micro-compression test on a microneedle patch comprised of an 11×11 array of microneedles. The microneedle patch tolerated compression force > 0.4 N/microneedle, which is sufficiently high to puncture the skin without causing the microneedles to mechanically yield (Fig.3a).²⁴ The microneedles penetrated the mouse skin, as evidenced by Trypan blue staining (Fig.3b). Hematoxylin and eosin (H&E) staining of the extracted mouse skin tissue further confirmed that the microneedles have penetrated the stratum corneum and perforated into the epidermal layer (Fig.3c, Supplementary Fig.9). The penetration depth is around 140–170 μm (Supplementary Fig.9). SEM images indicated that the microneedles maintained their conical shape and sharp tips after removal from the mouse skin, which further confirms their mechanical integrity during administration (Supplementary Fig.10). Furthermore, micro-indents on skin caused by the application of the microneedle patch quickly became invisible as the skin recovered to the normal state within 15 minutes (Supplementary Fig.11). The rapid recovery of the skin re-affirms the minimally-invasive nature of the procedure compared to conventional techniques such as blood collection.

Fig. 3 |. Biophysicochemical properties of microneedle patches.

a, Mechanical behavior for the microneedle patches under normal compressive load and schematic illustration of experimental setup (inset). b, Left: Mouse ventral skin administered with microneedle patch. Right: optical image of trypan blue staining showing the indents caused by the penetration of microneedle on mouse skin. Scale bar, 500 μm. c, H&E stained section of mouse skin showing the penetration of a single microneedle. Scale bar, 100 μm. d, Cell viability of human dermal fibroblast cells (HDF cells) co-cultured with pristine and BSA coated microneedle patch for 1 hour and 16 hours. Error bar represents standard deviation. N=3 repeated tests. e, H&E stained section of mouse organs with and without administration of microneedle patch, indicating excellent biocompatibility of microneedle patch. Scale bar, 200 μm.

Considering that the antibodies are immobilized on the microneedles, it is important to understand the stability of these proteins during the penetration of the microneedles into the dermal tissue, residence in the dermal tissue, and subsequent withdrawal (Supplementary Fig.12a). To test this, we coated microneedles with fluorescently labelled BSA (employed as a model protein) prior to application onto mouse skin for different durations (15 sec, 10, 20, 30 and 60 mins). Upon withdrawal, microneedles exhibited a ~20% loss in fluorescence intensity, indicating that a small fraction of proteins desorbed from the microneedles, possibly due to the shear forces between the microneedle surface and the epidermis (Supplementary Fig.12b). Considerably, the decrease in the fluorescence intensity (representing the amount of protein lost) did not depend on the residence time of the microneedles in the dermal tissue, indicating minimal loss from desorption or proteolytic degradation of these proteins in the dermal tissue, even for a residence time of 60 minutes.

To evaluate the biocompatibility of the polystyrene microneedle patches, human dermal fibroblasts (HDF), one of the major cell types in the dermis layer, were cultured in the presence of microneedles and BSA coated microneedles (since the in vivo sampling involves microneedles blocked with a dense layer of BSA). Notably, no change in cell viability was observed after either 1- or 16-hours of in situ culture with the microneedle patches (Fig.3d). Systemic toxicity of the microneedle patch was also investigated by H&E staining. Negligible changes in cell states between the control group and the microneedle administered group demonstrated the excellent biocompatibility of the microneedle (Fig.3e). In addition, we measured the endotoxin level of the microneedle patch to investigate the possibility of an unwanted immune response after administration. Microneedles coated with BSA exhibited negligible endotoxin levels compared to the limit set by the US Food & Drug Administration (20 EU/device) (Supplementary Fig.13, Supplementary Table.1). Taken together, these findings indicate that the biofunctionalized microneedle patch represents a safe approach for in vivo biodetection.

Detection of cocaine-specific antibody (IgG) in an immunized mouse model

Cocaine overdose and cocaine use disorder (CUD), which currently do not have FDA-approved medications, remain a global medical and social problem.^25,26 While vaccines are a unique approach that do not directly address the underlying neurobiological mechanism behind CUD, cocaine vaccines that produce antibodies reduce the rate and quantity of drug entry into the brain and inhibit the psychoactive effects of the drug.²⁷ Unlike other vaccines that confer prolonged protection, currently designed antidrug vaccines require frequent boosting to maintain the effective antibody levels. ^28–29 A recent study revealed that lack of pre-vaccination screening assays that predict the most effective vaccines or subjects amenable to vaccination is the major obstacle for clinical translation.³⁰ Therefore, technologies that enable rapid and reliable testing of hapten-specific antibody titers in vaccinated subjects may aid rational vaccine design and provide screening tools to predict vaccine clinical efficacy against drugs of abuse. Here, we set out to demonstrate a simple, rapid, and non-invasive method for evaluating the efficacy of the cocaine vaccine using a microneedle patch.

Mice were subcutaneously immunized and boosted with BSA-cocaine combined with adjuvants, lipopolysaccharide (LPS) and Alum, as depicted in Fig.4a. This vaccine was expected to result in the generation of two types of antibodies: anti-BSA and anti-cocaine. As a standard way to determine antibody titer and immunization efficacy, serum collected on the 7^th week was serially diluted and the concentrations of two types of antibodies (anti-BSA and anti-cocaine) were tested by the ELISA using BSA and BSA-cocaine as recognition elements, respectively (Fig.4b, c). Since BSA-cocaine is expected to bind with both types of antibodies simultaneously, the difference in the antibody concentrations obtained using BSA-cocaine and BSA represents the concentration of anti-cocaine antibody. Mice with higher anti-BSA-cocaine titer than the anti-BSA titer are considered as “vaccine responders” (Fig.4d, Supplementary Fig.14). These responder mice were subjected to microneedle administration to test the ability of this novel method to detect the presence of anti-cocaine antibody in ISF.

Fig. 4 |. Minimally-invasive detection of cocaine-specific antibodies in an immunized mouse model.

a, Schematic illustration showing the working principle of cocaine immunization, generation of BSA and cocaine-specific antibodies, and administration of the microneedle patch for the detection of specific antibodies in ISF. b, Schematic illustration demonstrating principle of anti-cocaine detection. There are two types of antibodies generated after cocaine immunization: anti-BSA and anti-cocaine. Since BSA-cocaine simultaneously bind to both types of antibodies, the difference in the antibody concentrations determined using BSA-cocaine and BSA coated plates represents the concentration of anti-cocaine antibody. c, Workflow of immunization, blood draw and microneedle administration on mice. d, Plot depicting log₁₀ titer of anti-BSA and anti BSA-cocaine in mice serum from both immunized and unimmunized group, tested by “gold standard” ELISA. N=5 mice in each group. Within unimmunized group, data statistically not significant (NS). P value > 0.9999 by one-way ANOVA with Tukey’s multiple-comparison test. Within immunized group, data statistically significant, **** P< 0.0001 by one-way ANOVA with Tukey’s multiple-comparison test.

To determine the shortest time scale for effective capture of antibodies, we administered the BSA-coated microneedle patch on the mouse dorsal skin for different durations. We found that 30 seconds of microneedle patch administration was sufficient to capture BSA-specific antibodies (Fig.4e). Longer administration of the microneedle patch did not significantly alter the fluorescence signals corresponding to the antibodies. To assess the site-to-site variations in the measured concentrations, we administered four microneedle patches coated with BSA on the ventral and on the dorsal skin of a responder mouse. The microneedle patches were subsequently probed by anti-mouse IgG and plasmonic-fluor ex vivo. We noted very small differences (relative standard deviation < 8%) in the fluorescence intensity among the four microneedle patches administered on the same side of the mouse. However, the four microneedle patches administered on the ventral side exhibited nearly 30–40% higher fluorescence intensity compared to those on the dorsal side (Fig.4f). This difference possibly stems from the differences in the thickness of skin and blood flow between ventral and dorsal sides of the mice.^31–32 This observation underscores the importance of accounting for site-to-site variation and consistent administration of the microneedle patches to ensure reliable comparison across different subjects and experiments.

To validate the applicability of the plasmonic-fluor enhanced microneedle patch in detecting cocaine-specific antibodies in a minimally-invasive manner, we employed five unimmunized mice and five responder-mice. Each mouse was administered with two microneedle patches coated with BSA and BSA-cocaine, and the patches were left on the skin for only 30 seconds. In the five responder-mice, we observed high fluorescence signal and significant difference in the fluorescence intensity between the BSA and BSA-cocaine microneedle patches. On the other hand, in the case of the microneedle patches from five unimmunized mice, we noted a much lower fluorescence signal and negligible difference between the BSA and BSA-cocaine microneedle patches (Fig.4g). Taken together, these results indicate that the plasmonic-fluor enhanced microneedle patch is a promising tool to determine and evaluate the vaccine response efficiency in a simple and non-invasive manner. In contrast to conventional blood-drawing, the microneedle method enables frequent and easy monitoring of vaccine efficiency in individuals and can potentially accelerate vaccine development.

Detection and quantification of cytokines in an endotoxin-induced shock mouse model

Next, we set out to demonstrate the detection of cytokines in dermal ISF of an LPS-induced endotoxin shock mouse model using the microneedle patch. LPS, a pathogen-associated molecular pattern (PAMP), triggers innate immunity and induces secretion of multiple pro-inflammatory cytokines (Fig.5a). Measurement and monitoring of these pro-inflammatory cytokines in blood has been an established method for evaluating the ability of immune system to mount an innate inflammatory immune response.³³

Fig. 5 |. Longitudinal monitoring and quantification of cytokines in an endotoxin shock mouse model.

a, Schematic illustration showing the working principle of immune response induced by LPS administration, cytokine generation and administration of the microneedle patch for longitudinal detection. b, Timeline of endotoxic shock (LPS injection), longitudinal microneedle administration on mice, and blood collection (for validation of the microneedle test). c, Fluorescence map representing mouse IL-6 captured on the microneedles before and at 1, 2.5 and 4 hours post LPS injection. Scale bar, 200 μm. d, Plot depicting concentrations of mouse IL-6 in ISF before and at 1, 2.5 and 4 hours post LPS/saline injection measured using microneedle. (n=3 in each group) Data statistically significant, **** P< 0.0001 by two-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test. e, Plots showing the comparison of concentrations of IL-6 measured in serum and ISF in saline group (left) and LPS group (right). (n=3 in each group) At 4 hours post LPS injection, IL-6 in dermal ISF determined by plasmonic-fluor enhanced microneedle (blue) exhibited good qualitative correlation with that in serum tested by conventional ELISA (grey). Error bars represent standard deviation. Error bars of serum results are technical repeats.

BALB/C mice were injected intra-peritoneally with LPS (1 μg/g) to induce an acute phase response and systemic inflammation. Using functionalized microneedle patches, we measured the longitudinal concentrations of mouse interleukin-6 (IL-6) in ISF, which is known to increase in serum over 4 hours after LPS administration.³⁴ Microneedle patches, pre-functionalized with IL-6 capture antibodies, were administered at different time points on mouse ventral skin and left undisturbed for 20 minutes (as indicated in Fig.5b). After removing the microneedle patches from the skin, p-FLISA was performed ex vivo to measure the concentration of IL-6. Fluorescence intensity corresponding to the plasmonic-fluors on the microneedle patches exhibited a gradual increase from 1 to 4 hours post LPS injection (Fig.5c), while no significant change was observed in mice injected with saline (negative control group) (Fig.5c, Supplementary Fig.15). Based on the standard curve (obtained using microneedle patches exposed to known concentrations of IL-6), the concentrations of IL-6 in mouse ISF were determined to be equivalent to 2.6±1.9, 12.3±8.6, 120.4±73.4 and 1271.9±393.4 pg/ml at 0, 1, 2.5 and 4 hours post LPS injection, respectively (Fig.5d). Specificity tests also validated that other cytokines or chemokines, whose levels might also increase with LPS injection, do not interfere with the detection and quantification of IL-6 on microneedle (Supplementary Fig.17, Supplementary Table.2). The IL-6 concentration in sera collected 4 hours after LPS/saline injection exhibited good qualitative correlation with that measured in ISF using the microneedle method at the same time point (Fig.5e). However, the serum IL-6 concentration was found to be nearly 22-fold higher compared to that in the ISF. The absolute concentration of the protein biomarkers in ISF is lower than the concentration in blood, which can partly be ascribed to the difference between microneedle-based analyte sampling method and solution-based standard curve, as well as inherent variation of proteins in body fluids.⁴ The p-FLISA standard curve, which is used for estimating the concentration of the analyte, is obtained by exposing the microneedle patches to known concentrations of IL-6 in standard dilution buffer. In contrast, microneedle based ISF sampling occurs in a “dense tissue matrix”, resulting in slower diffusion kinetics and consequently a lower “apparent concentration” of the analyte. Nevertheless, the analyte concentration determined using the microneedle-based method also exhibited excellent qualitative agreement with the measured concentrations in serum samples (Fig.5e, Supplementary Fig.16).

Frequent and timely measurement of protein biomarkers is critical for disease monitoring and diagnostics in both biomedical research and clinical applications. Unfortunately, conventional longitudinal measurements require frequent blood draws in a short period, which may cause iatrogenic anemia and elevate morbidity of patients. Moreover, it is often impossible to repeatedly draw blood from small experimental animals, which will result in their death. The minimally-invasive microneedle method represents a transformative approach to perform frequent, sensitive, and accurate measurements of protein biomarkers in a longitudinal manner in the same mouse.

As opposed to in vitro diagnostics that involve sample acquisition (such as blood, urine) at a specific timepoint along the disease progression and analysis at a later point in time, in vivo diagnostics involving capture of target analytes from a dynamically varying matrix (e.g., dermal ISF in the present case) is inherently a non-equilibrium condition. This is particularly true for the cases in which the target analyte concentration varies within the sampling timescales, for example, IL-6 level in LPS-stimulated mice. In such cases, the concentration determined using microneedle patch represents a time-average concentration of the analyte in ISF over the sampling period. From a diagnostic translation standpoint, this time-averaged concentration can be standardized by setting rigorous guidelines for microneedle administration (e.g., administration time, location) and ex vivo analysis (e.g., standard curve conditions).

Detection and quantification of endogenous matricellular protein in periosteum

The quantification of endogenous biomarkers at specific sites or inner tissues of interest is highly desirable for biomedical and clinical research. Conventional biofluids, such as blood, are not able to reflect local concentrations of biomarkers. Furthermore, such local detection and monitoring of relevant biomolecules remains challenging due to the difficulty in collecting an adequate amount of biofluid in a minimally invasive manner. While invasive procedures are sometimes inevitable for detection of protein biomarkers in inner tissues or organs, the microneedle-assisted technique will be able to minimize the damage to the target sites and allow repeated sampling. Here we hypothesized that the microneedle patch would efficiently sample and measure protein analytes at specific tissue or membrane locations with high sensitivity and specificity in a minimally-destructive manner (Fig.6a).

Fig. 6 |. Detection of periostin in periosteum by microneedle patch.

Endogenous matricellular periostin was detected in calvarial periosteum and skin of the wild type (WT) mice but not the periostin knockout (Postn^KO) mice with microneedle patch. a, Schematic illustration showing the microneedle approach and conventional approach to measure the endogenous biomarkers in calvarial periosteum. Microneedle detection is minimally-destructive, and mice can recover after testing; standard analysis requires scraping of the skull and isolation of periosteum, which usually necessitates euthanasia. b, Images of histology (H&E) and immunohistochemistry (IHC) showing a high local concentration of the periostin. The calvaria of WT and Postn^KO mice were immunostained with antibodies against periostin (brown). Periosteum (top, arrowheads) of WT mice exhibit strong expression of periostin, while staining was absent in WT sections lacking primary antibody (secondary antibody only control) and in sections of calvaria from Postn^KO mice. Across the surface of skull, the average thickness of the calvarial periosteum was 29+/−15 um. B represents the position of bone. c, Optical images showing the administration of the microneedle patch on mouse calvarial periosteum. Head of mice was stabilized on a stereotaxic instrument. d, Plot depicting concentration of periostin on skin and periosteum of the WT and Postn^KO mice determined by microneedle patches. Both periosteum and skin of WT mice (n=2) demonstrate high concentration of periostin, while Postn^KO mice (n=2) show negligible amount. Error bars represent standard deviation. Data statistically significant P value = 0.0206 for microneedle on periosteum and P value = 0.0153 for microneedle on skin, * P< 0.1 by one-tailed unpaired t-test with Welch’s correction. e, Plot showing concentration of periostin in serum of the WT and Postn^KO mice. Serum of WT mice reveal high concentration of periostin, while Postn^KO mice have a negligible amount. Error bars represent standard deviation. Data statistically significant P value = 0.0205, * P< 0.1 by one-tailed unpaired t-test with Welch’s correction.

The periosteum is a fibro-cellular membrane, which covers the outer surface of bones and plays key roles in bone growth, fracture healing and skeletal regeneration.^35–36 In rodents and humans, the average thickness of the periosteum is 40 μm and 100 μm for both tibia and femur, respectively.^37–38 In our study, in the mouse calvaria the average periosteal thickness was 29±15 μm (Fig.6b). The extracellular matrix of the periosteum contains a high local concentration of the matricellular protein, periostin. It is encoded by Postn gene and plays important roles in bone regeneration and bone tumor metastasis.^39–40 In addition, a circulating isoform of periostin has been identified as a potential biomarker of bone density loss and osteoporosis, tumor metastasis, and airway disease.^41–42 Herein, we set out to test the applicability of our microneedle patch for the detection and quantification of periostin in a novel tissue, the calvarial periosteum, and in the dermal ISF.

We employed transgenic periostin knock-out mice (Postn^KO) and wild type mice (WT) as experimental and control groups, respectively. WT mice demonstrated high expression of POSTN protein in the calvarial periosteum by immunohistochemistry (IHC), which was absent in Postn^KO mice (Fig.6b). To detect periostin, microneedles were pre-functionalized with anti-periostin antibodies and administered to the calvarial periosteum layer for 10 minutes (Fig.6c). Simultaneously, another pre-functionalized patch was administered on the dorsal skin of the same mouse for 10 minutes. Subsequently, p-FLISA assay was performed ex vivo to measure the local concentrations of periostin. Fluorescence intensity corresponding to the plasmonic-fluors on the microneedle patches (both on periosteum and dorsal skin) exhibited a strong signal from WT mice, while no significant signal was observed in Postn^KO mice (Fig.6d). Based on the standard curve, concentrations of periostin in periosteum and skin were around 2000 pg/ml from WT, as compared to negligible amount in transgenic Postn^KO mice.

Compared to conventional methods, the plasmonic-fluor enhanced microneedle method did not require extraction or isolation of periosteum during analysis, indicating that multi-round testing and long-term monitoring is achievable (Fig.6a). It is of note that the concentration of periostin in periosteum and skin measured by microneedle is nearly 450-fold and 330-fold lower than the concentration in mouse serum, which was around 950 ng/ml (Fig.6e). This phenomenon is possibly due to binding and retention of periostin within the extracellular matrix, reducing the amount of available perostin which can be captured by the microneedle. Since the sampling area for skin and periosteum is different, absolute comparison of the values between these two tissues is precluded. It is also worth mentioning that blood periostin represents a systemic level of periostin while local periostin plays important roles in stem cell recruitment. Understanding its specific level is critical especially when there is a change introduced to the local environment of periosteum/bone. For example, if fracture occurred on the bone and the regeneration process has started, the local level of periostin at defined locations around this fracture can be revealed without significantly disturbing the regeneration process. In this way, it is easier to track the progress of bone regeneration and better understand how periostin contributes to this process by looking at changes in local periostin levels. The same concept could be applied in diverse fields to analyze desired target biomarkers and understand disease progression. In general, these results indicate that microneedle patch can be used to evaluate the amount of an endogenous matricellular protein biomarker at specific tissue location in a minimally-invasive manner.

Discussion

In summary, we have designed a minimally-invasive, ultrasensitive and quantitative biodetection technology based on a bilayered microneedle patch with plasmonic-enhanced fluoroimmunoassay. Through a series of mouse models, we demonstrated that this novel technology enables both simple and timely detection of biomarkers of interest including longitudinal monitoring of inflammatory immune responses, evaluation of vaccine efficiency in a minimally-invasive manner, as well as quantification of localized changes protein content within the tissue microenvironment. While largely retaining or enhancing the sensitivity and convenient workflow of conventional immunoassays, the plasmonic-enhanced microneedle method overcomes the need for tedious sample collection (e.g., blood draw, ISF extraction), making it highly attractive for clinical diagnostics. Although we focused on the detection of antibodies, cytokines and matricellular proteins in this work, the novel biodetection method can be broadly applicable to a wide range of other diseases that can be effectively intervened by rapid and timely diagnosis, as well as by personalized treatment. For example, the diagnosis of myocardial infarction can be expedited by applying the microneedle patch (specific to cardiac troponin) in the ambulance as the patient is being moved to an emergency room after experiencing chest pain. Moreover, this technology could enable at-home sampling and centralized detection of the biomarker levels through self-administration of microneedle patch, which can ultimately enable timely monitoring of therapy, such as certain antibiotic treatments, chemotherapy or checkpoint inhibitor immunotherapy. Therefore, this novel sensing technique can potentially be employed in point-of-care and resource limited settings, to facilitate rapid disease diagnosis and efficient therapeutic intervention in a more patient-friendly manner.

We note that various protein biomarker levels in the ISF are not well characterized and understood, which is an important bottleneck in the clinical translation of this technology.⁴ Extensive efforts need to be dedicated to understanding the biomarker levels in ISF, their correlation with blood and, more importantly, the associated lag time correlated with blood, before this technology could be employed in clinical diagnostics. The technology demonstrated in this manuscript serves as an efficient tool for biomedical and clinical researchers to fill this knowledge gap and propel the technology to clinical applications.

Methods

Animals:

All procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) at Washington University in St. Louis. Mice were housed in the housing facility at a constant temperature (21–23 °C) and humidity (45–50%) on a 12 hours light-dark cycle (lights on 0700–1900 hr), with food and water available ad libitum throughout the studies.

Synthesis of magnetic nanoparticles:

Magnetic nanoparticles (Fe₃O₄ NPs) were synthesized via previously reported polyol synthesis method.⁴³ Briefly, 0.15 g of iron (III) chloride hexahydrate (Sigma Aldrich, 236489) was dissolved in a mixture of 3 mL ethylene glycol (Sigma Aldrich, 102466) and 1 mL ethanolamine (Sigma Aldrich, 398136) to form a stable light brown solution. After 30 minutes, 167.5 mg of polyethylene glycol (Sigma Aldrich, P3640) and 663.6 mg of sodium acetate trihydrate (Sigma Aldrich, S8625) was added under vigorous stirring. Subsequently, the solution was transferred to a Teflon-lined stainless-steel autoclave and heated to 200°C for 8 hours. The Fe₃O₄ NPs were collected and washed for three times, with ethanol and water, and re-dispersed in ethanol for further use.

Fabrication of microneedle:

Microneedles were prepared using silicone molds with conical holes (Blueacre Technology Ltd.). Each microneedle is 600 μm in length with a diameter of 300 μm at the base. The center-to-center spacing between the microneedles is 600 μm. Polystyrene/dichloromethane solution (25% w/v) was deposited on the silicone mold and left under vacuum at the bottom to create a negative pressure, followed by drying at room temperature for at least 4 hours, allowing the evaporation of the solvent and the formation of the polystyrene microneedle. Fe₃O₄ nanoparticles dispersed in polystyrene/dichloromethane solution (final concentration: 15% w/v of polystyrene and 8 mg/ml Fe₃O₄ nanoparticle in dichloromethane) were deposited on the back of the polystyrene microneedle patch, followed by vacuum condition. After drying in room temperature for 4 hours, microneedle patch was carefully separated from the mold and tailored into small pieces with square shape for further use. The preparation and storage of microneedle patches were performed in sterilized condition.

Synthesis of plasmonic-fluor:

Synthesis of plasmonic-fluor was performed according to a procedure described in our previous report.⁴⁴

Synthesis of AuNR:

Wavelength of gold nanorods can be easily tuned to couple with dye molecules in order to achieve best enhancement factor.⁴⁵ To prepare plasmonic-fluor-800CW, AuNR-760 (LSPR wavelength ~760 nm) was prepared by seed-mediated method.^46,47 To prepare seed solution, 0.6 ml of 10 mM ice-cold NaBH₄ solution (Sigma Aldrich, 71321) was added into a solution containing 9.75 ml 0.1 M CTAB (Sigma Aldrich, H5882) and 0.25 ml 10 mM HAuCl₄ (Sigma Aldrich, 520918) under vigorous stirring at room temperature for 10 min. The solution changed from yellow to brown which indicates the formation of Au seed. To synthesize gold nanorods, the growth solution was prepared by the sequential addition of 2 ml 0.01 M HAuCl₄ aqueous solution, 38 ml 0.1 M CTAB, 0.55 ml 0.01 M AgNO₃ (Sigma Aldrich, 20439 0), 0.8 ml 1 M HCI (Sigma Aldrich, H9892) and 0.22 ml 0.1 M ascorbic acid (Sigma Aldrich, A92902) followed by gentle homogenization. Subsequently, 5 μl of the seed solution was added into the growth solution and left undisturbed in dark for 24 hours. AuNR solution was collected by centrifugation at 6000 rpm for 40 minutes to remove the supernatant. AuNR was then re-dispersed into nanopure water for further use.

Conjugation procedures:

Bovine serum albumin (BSA) was first conjugated with biotin and 800CW sequentially through EDC/NHS chemistry. Specifically, 2 mg NHS-PEG4-biotin (Thermo Scientific, prod number 21329) was added to 2.2 ml 5 mg/ml BSA (Sigma-Aldrich, A7030) in 1X PBS and incubated at room temperature for 1 hour. BSA-biotin conjugation was purified by a desalting column (Thermo Scientific, Prod number 21329, 7000 MWCO). Next, 800CW was conjugated to BSA-biotin. 0.1 ml 1 M potassium phosphate buffer (K₂HPO₄, pH=9) was added into 1 ml purified BSA-biotin solution to raise the pH. Next, 25 μl 4 mg/ml NHS-800CW (Licor, P/N 929–70020) was added to the mixture and the solution was incubated at room temperature for 2.5 hours. BSA-biotin-800CW was purified by Zeba desalting column pre-equilibrated with nanopure water.

Synthesis of plasmonic-fluor:

To prepare plasmonic-fluor-800CW, AuNR (wavelength around 760) was employed as the nanoantenna. 1 μl MPTMS (Sigma Aldrich, 175617) was added to 1 ml AuNR (extinction ~2) and the mixture was shaken on rocking bed for 1 hour. Subsequently, MPTMS-modified AuNR was collected by centrifugation at 6000 rpm for 10 mins and was further mixed with 2 μl APTMS (Sigma Aldrich, 281778) and 2 μl TMPS (Sigma Aldrich, 662275) to form the polymer spacer layer. Finally, AuNR/polymer solution was collected by twice centrifugation at 6000 rpm for 10 minutes to remove the free monomer and concentrated into a final volume of 10 μl.

Next, BSA-biotin-800CW conjugate was coated around AuNR/polymer modified from a previously reported method.⁴⁸ Specifically, pH of 100 μl 4 mg/ml BSA-biotin-800CW was first lower by adding 1 μl 20 mg/ml citric acid (Alfa Aesar, 36664). Subsequently, concentrated AuNR/polymer solution was added into BSA-biotin-800CW solution and sonicated for 20 minutes under dark condition. The coated nanostructures were then collected by centrifugation at 5000 rpm for 5 minutes and subsequently incubated with 0.5 ml 0.4 mg/ml BSA-biotin-800CW (pH=10) for 3 days in 4°C. Finally, the nanostructures were washed four times using alkaline nanopure water (pH=10) by centrifugation at 6000 rpm and re-dispersed in 1% BSA 1X PBS solution for further use.

Fluorescence enhancement using plasmonic-fluor:

The test procedure is schematically illustrated in Supplementary Fig.7. Specifically, BSA-biotin was first immobilized on 96-well plate by incubating the well with 50 ng/ml BSA-biotin in 1X PBS at room temperature for 15 minutes. The plate was washed by three times using PBST (0.05% Tween 20 in 1X PBS) and then blocked using Odyssey® Blocking Buffer (PBS) (Licor, P/N 927–40100). 1 μg/ml streptavidin-800CW was subsequently added and incubated for 10 minutes. Next, the plate was washed three times using PBST and then incubated with ~76 pM plasmonic-fluor-800CW (in 1% BSA). Finally, after washing, 200 μl of PBST was added into each well and the fluorescence signal before and after the addition of plasmonic-fluor was recorded using Licor CLX fluorescence imager with the following scanning parameters: laser power~L2; resolution~169 μm; channel: 800; height: 4 mm.

Material characterization:

Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100F field emission (FE) instrument. A drop of aqueous solution was dried on a carbon-coated grid, which had been made hydrophilic by glow discharge. SEM images were obtained using a FEI Nova 2300 field‐emission scanning electron microscope at an acceleration voltage of 10 kV. The extinction spectra of plasmonic nanostructures were obtained using a Shimadzu UV-1800 spectrophotometer. Fluorescence mappings were recorded using LI-COR Odyssey CLx imaging system. The X-ray diffraction (XRD) patterns of the Fe₃O₄ nanoparticles were obtained using a Bruker D8-Advance X-ray powder diffractometer using Cu Kα radiation (λ = 1.5406 Å) over the 2θ range 10^o – 90^o.

Mechanical test:

The mechanical properties of the microneedle patch were measured by displacement-force test station (Instron 5583 electro-mechanical Universal Testing Machine) (Fig.3a). A microneedle patch was attached to a rigid platform with microneedles facing up. The sensor probe was brought in contact with the microneedles in the vertical direction at a speed of 0.1 mm s⁻¹. The initial distance between the sensor and microneedle tips was set to be 1 cm. Displacement-force measurements were acquired from the point at which the sensor first touched the microneedle tips to the onset of buckling of the microneedles.

Skin penetration efficiency test:

Mouse skin after administration of microneedle patch was imaged by digital camera to assess skin penetration efficiency. After administration of microneedle patch for 15 minutes, mouse ventral skin was stained with trypan blue for 20 minutes. Mouse was subsequently euthanized, and the skin sample was imaged by digital camera after wiping remaining dye from the skin. In a separate experiment, after 15 minutes of administration of microneedle patch, mouse was euthanized, and its ventral skin was isolated carefully. These excised skins were washed with deionized water and then fixed with 4% formalin solution to stain with hematoxylin and eosin (H&E). Histological examination was conducted by an optical microscope under bright field illumination (Biotek Lionheart FX).

Protein retention test:

To access stability of protein bound on the microneedles, the microneedles were coated with BSA-CW800. After washing with PBST and drying, fluorescence images were recorded using LI-COR Odyssey CLx imaging system. Subsequently, microneedles were administered on mouse dorsal skin and left for different periods of time, varying from 15 seconds to 60 minutes. Fluorescence images were recorded again using the same parameters in LI-COR Odyssey CLx imaging system. The fluorescence intensity before and after administration was compared to assess the retained fraction of the protein on the microneedle surface.

Assessing in vitro biocompatibility of microneedle patch:

To evaluate toxicity of microneedle in vitro, human dermal fibroblast cells (HDF) were selected as a model system. Pristine microneedle and BSA coated microneedle were incubated in cell culture medium for 1 hour and 16 hours, representing short-term and potentially long-term contact with skin tissues. After removing microneedle patch, cell culture medium was employed to incubate with HDF for 24 hours. The cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.

Assessing systemic toxicity of microneedle patch:

For toxicity assessment in vivo, mice administered with BSA-coated microneedle patch were selected as the treatment group, while mice without administration were used as control group. The representative organs including heart, lung, liver, kidney and spleen in each group were harvested and fixed in 4% neutral buffered formalin for histological analysis, which were subsequently processed by The Musculoskeletal Histology and Morphometry Core at Washington University in St. Louis for paraffin embedding, sectioning, and hematoxylin and eosin staining (H&E staining). Histological examination was conducted by an optical microscope under bright field illumination (Biotek Lionheart FX).

Bacterial endotoxin level of microneedle patch:

To evaluate bacterial endotoxin level on microneedle surface, especially after blocking with BSA, microneedle patch coated with BSA were first incubated with endotoxin-free water overnight. Subsequently, the bacterial endotoxin level inside was detected and measured by Kinetic Chromogenic Limulus Amebocyte Lysate (LAL) assay (Thermo Scientific, Catalog number # 88282).

Mouse IL-6 ELISA on microneedle and on microtiter plate:

Mouse IL-6 DuoSet ELISA kit (R&D systems, catalog number DY406, lot number P195781) was employed in the study. As-prepared magnetic microneedle patches were first placed in a 24 well plate (Corning, PART# 3526) which was clamped on a magnetic plate separator (Luminex Corporation, PART# CN-0269–01) designed to accommodate the microplate. Microneedle patches were immersed and incubated with 1 ml of capture antibodies (2 μg/ml in PBS, R&D systems, PART# 840171) through overnight incubation at room temperature, followed by washing and blocking with 2 ml reagent diluent (1X PBS containing 3% BSA, 0.2 μm filtered). During incubation and washing steps, microneedle patches were tightly attached to the bottom of well. After three times washing with PBST, 1 ml of serial diluted standard samples (R&D systems, PART# 840173) were added into different wells and microneedle patches were incubated at room temperature for 2 hours. Subsequently, patches were washed and incubated with biotinylated detection antibodies (R&D systems, PART# 840172, 75 ng/ml in reagent diluent) for 2 hours, washed again with PBST, and incubated with HRP-labeled streptavidin (R&D systems, PART# 893975, 40-fold dilution using reagent diluent) for 20 mins. 1 ml of substrate solution (1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (tetramethylbenzidine) (R&D Systems, Catalog # DY999)) was added to each well and the reaction was stopped by adding 500 μl of H₂SO₄ (2 N) (R&D Systems, Catalog # DY994) after 20 mins. Optical density of each well was determined immediately using a microplate reader set to 450 nm, after removing microneedle patch from each well. IL-6 ELISA on 96 well plate was implemented in the same manner.

Mouse IL-6 FLISA and p-FLISA on microneedle:

Mouse IL-6 FLISA was implemented adopting the similar approach as the ELISA described above, except that HRP-labeled streptavidin was replaced by 800CW-labeled streptavidin (LI-COR P/N 926–32230, 20 ng/ml for 20 minutes). The patches were washed three times each using PBST followed by nanopure water. In case of p-FLISA, 1 ml plasmonic-fluor-800CW was added subsequently (extinction ~0.5), incubated for 30 minutes, and the patches were washed 3 times each with reagent diluent followed by PBST. Patches were imaged using Licor CLx fluorescence imager with the following scanning parameters: laser power~L2; resolution~21 μm; channel: 800; height: 0 mm.

Detection of cocaine-specific antibody (IgG) in an immunized mouse model:

Animals and cocaine immunization:

Male mice (C57BL/6, Jackson Lab # 000664), at the age of 5–6 weeks, were purchased from Jackson Labs (Bar Harbor, ME, USA). Mice were housed four per cage and allowed to acclimate for 7 days before the experiment in the housing facility. Eleven mice were randomly divided into two groups, vaccination group and control group. For each mouse in vaccination group, cocaine vaccination solution including 100 μg cocaine-BSA conjugate (Fitzgerald Industries, PART# 80–1037) and 2 μg lipopolysaccharide (InvivoGen, catalog code: vac-3pelps) in 100 μl saline mixed with 100 μl 2% Alhydrogel (InvivoGen, catalog code: vac-alu-250) was freshly prepared prior to vaccination. Each mouse was first vaccinated subcutaneously with totally 200 μl of cocaine vaccine solution on four injection sites and boosted with half total dosage on two sites at day 14, 21 and 28, both on the dorsal side. Two weeks post last boost, blood from both vaccinated and control group was collected via the submandibular vein and serum was stored at −20 °C.

Detection of anti-cocaine antibody in mouse serum:

96-well ELISA plates (Thermo Scientific, Catalog # 15041) were coated with 1 μg/ml BSA or cocaine-BSA conjugate in PBS at 4°C overnight and blocked with 300 μl of Odyssey® Blocking Buffer for 1 hour. Serum was diluted in PBST at a range of dilution in duplicates and applied on both BSA and cocaine-BSA conjugate coated wells for 1 hour at room temperature. After washing with PBST, plate was incubated with a biotin labeled donkey anti-mouse antibody (R&D systems, Catalog # BAF018, 1:2000 in 1% BSA-PBST, 100 μl per well) for another 1 hour, followed by HRP-labeled streptavidin (R&D systems, PART# 893975, 40-fold dilution) for 20 mins. 100 μl of substrate solution (1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (tetramethylbenzidine) (R&D Systems, Catalog # DY999)) was added to each well and the reaction was stopped by 50 μl of H₂SO₄ (2 N) (R&D Systems, Catalog # DY994) and absorbance measured at 450 nm. Within vaccination group, anti-cocaine-BSA titer of each mouse was defined by three times of standard deviation plus its mean anti-BSA titer. Mice with higher anti-cocaine-BSA antibody titer than the anti-BSA titer were defined as “vaccine responders”.

In situ sampling and quantification of cocaine specific antibody in mouse dermal ISF through microneedle:

Studying effect of administration time antibody detection:

Microneedle patches were incubated with BSA (1 μg/ml in PBS) in a 24-well plate at room temperature overnight, followed by washing and blocking with Odyssey® Blocking Buffer for 1 hour. After three times washing with PBST and blow drying with nitrogen gas, microneedle patches were administered on dorsal skin of mouse (vaccine responder) under anesthesia. The period of administration varied from 30 seconds to 5 minutes. Subsequently, patches were washed with PBST and blocked by Odyssey® Blocking Buffer for another 30 minutes, followed by incubation with biotin labeled donkey anti-mouse antibody (1:2000 in 1% BSA-PBST) for 1 hour and 800CW-labeled streptavidin (20 ng/ml) for 20 minutes. 1 ml Plasmonic-fluor-800CW (extinction ~0.5) was added subsequently, incubated for 30 minutes, and the patches were washed 3 times each with PBST. Patches were imaged using Licor CLx fluorescence imager with the following scanning parameters: laser power~L2; resolution~21 μm; channel: 800; height: 0 mm.

Studying effect of administration location for antibody detection:

Eight microneedle patches were incubated with BSA (1 μg/ml in PBS) in a 24-well plate at room temperature overnight, followed by washing and blocking with Odyssey® Blocking Buffer for 1 hour. After three times washing with PBST and blow drying with nitrogen gas, four microneedle patches were administered on dorsal skin and another four patches were administered on ventral skin of mice for 30 seconds, simultaneously. Subsequently, patches were washed with PBST and blocked by Odyssey® Blocking Buffer for another 30 minutes, followed by incubation with biotin labeled donkey anti-mouse antibody (1:2000 in 1% BSA-PBST) for 1 hour and 800CW-labeled streptavidin (20 ng/ml) for 20 minutes. 1 ml Plasmonic-fluor-800CW (extinction ~0.5) was added subsequently, incubated for 30 minutes, and the patches were washed 3 times each with PBST. Patches were imaged using Licor CLx fluorescence imager with the following scanning parameters: laser power~L2; resolution~21 μm; channel: 800; height: 0 mm. Intensity of fluorescence signal represents the amount of anti-BSA antibody binding to the BSA on microneedle surface.

Detection of anti-cocaine antibody through microneedle:

Microneedle patches were incubated with BSA or BSA-cocaine conjugate (1 μg/ml in PBS) in a 24-well plate at room temperature overnight, followed by washing and blocking with Odyssey® Blocking Buffer for 1 hour. After washing and drying, two microneedle patches coated with BSA and BSA-cocaine conjugate were simultaneously administered on dorsal skin of mouse under anesthesia for 30 seconds. Subsequently, microneedle patches were washed and blocked, followed by incubation with anti-mouse antibody for 1 hour and 800CW-labeled streptavidin for 20 minutes. 1 ml plasmonic-fluor-800CW was added subsequently (extinction ~0.5) and incubated for 1 hour. Patches were imaged using LICOR CLx fluorescence imager using the same scanning parameters as stated above.

Detection and quantification of cytokines in an endotoxin shock mouse model:

Animals and induction of endotoxin shock:

Female mice (BALB/C, Jackson Lab # 000651), at the age of 5–6 weeks, were purchased from Jackson Lab (Bar Harbor, ME, USA). Mice were housed three per cage and allowed to acclimate for 7 days in the housing facility before the microneedle experiment. To induce endotoxin shock, mice received intraperitoneal (i.p.) injection of lipopolysaccharide (InvivoGen, catalog code: vac-3pelps, 1 mg/kg mouse), while in control group mice received i.p. injection of saline solution.

In situ sampling and quantification of mouse IL-6 in dermal ISF through plasmonic-fluor enhanced microneedle:

To sample IL-6 in mouse dermal ISF, microneedle patches were pre-functionalized with IL-6 capture antibody, followed by washing with PBST and blocking with reagent diluent (1X PBS containing 3% BSA, 0.2 μm filtered) for 1 hour. Microneedle patches were administered on mice ventral skin under anesthesia for 20 minutes for obtaining baseline IL-6 measurements. Subsequently, mice were injected with LPS and microneedle patches were administered on mice at 1, 2.5 and 4 hours after injection in a successive manner. Followed by additional 30 minutes blocking of reagent diluent (1X PBS containing 3% BSA, 0.2 μm filtered), microneedle patches were exposed to biotinylated detection antibody, streptavidin-800CW and plasmonic-fluor as described above. Fluorescence images of microneedle patches were obtained using LICOR CLx fluorescence imager using the same scanning parameters described above. Blood was collected via the submandibular vein right after the removal of microneedle patch at last timepoint (4 h post LPS injection) and serum was stored at −20 °C for further analysis. IL-6 ELISA was performed as previously described on 96 well microtiter plate to determine the concentration of IL-6 in mouse serum.

To investigate the relationship between serum and ISF at early time points, we have employed another three groups of mice (randomly divided from 9 mice), administered microneedle patches and collected blood at earlier timepoints, namely before LPS injection, 1 and, 2.5 hours post LPS injection, respectively. Blood was collected via the submandibular vein right after the removal of microneedle patch at each timepoint and serum was stored at −20 °C for further analysis. Methods and reagents to determine the concentration of IL-6 in ISF and serum are identical to those mentioned in previous section. Based on the standard curve, concentration of IL-6 in mouse dermal ISF exhibited a gradual increase from pre-LPS injection state to 2.5 hours post LPS injection. The IL-6 concentration in sera also exhibited a similar increase in concentration from pre-LPS injection state to 2.5 hours post LPS injection, which is in agreement with that observed using microneedle method (Supplementary Fig.16).

Specificity of the IL-6 test:

It is possible that the fluorescence signal on microneedle is generated by other cytokines or chemokines whose concentration also increase post LPS injection. To investigate the specificity of the test, we incubated different concentration of human IL-6, mouse IFN-γ, mouse TNF-α, mouse IL-12, mouse CXCL9, mouse CCL19 and mouse CCL20 with mouse IL-6 capture antibody, followed by mouse IL-6 detection antibody and HRP/plasmonic-fluor (concentration and source of each protein were listed in Table S2). For conventional plate-based ELISA, p-FLISA and microneedle-based p-FLISA, assay method and reagents are identical to those mentioned in the previous section.

ELISA (Supplementary Fig.17a) and p-FLISA (Supplementary Fig.17b) implemented in microtiter plate indicate that other mouse cytokines or chemokines and IL-6 from other species do not interfere with the quantification of mouse IL-6. Different concentrations of these interfering proteins exhibited signals comparable to that of the blank. In addition, p-FLISA performed on microneedle (Supplementary Fig.17c) also exhibited weak signals (similar or lower to the blank) corresponding to interference proteins. (Please note that the Y axis in Supplementary Fig.17c is in logarithmic scale.)

Detection and quantification of endogenous matricellular protein periostin in periosteum and skin Animals:

Periostin knockout mice and wild type littermate controls on a mixed B6;129 background (Postn^tm1Jmol, Jackson Labs # 009067) were a gift from Dr. Muhammad Farooq Rai (Department of Orthopedic Surgery, Washington University in St. Louis). Two wild type (WT) male and two periostin knockout (Postn^KO) male mice were used for the plasmonic-fluor enhanced microneedle detection of periostin at the age of 12 weeks.

Histology and Immunohistochemistry:

The calvaria of wild type (WT) and periostin knockout (Postn^KO) mice were processed by Musculoskeletal Histology and Morphometry Core at Washington University in St. Louis for paraffin embedding, sectioning, and hematoxylin and eosin staining (H&E staining). Unstained tissue slides were acquired from the core for periostin immunostaining. Briefly, antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 50–55°C overnight. The tissues were then permeabilized in 0.2% Triton X-100 buffer (Sigma-Aldrich 9002–93-1) for 10 minutes, blocked in 2.5% normal horse serum (IMMPRESS HRP Anti-Rabbit IgG kit, Vector Laboratories, MP-7401) for 1 hour at room temperature, and incubated with anti-periostin antibody (Sigma-Aldrich HPA012306) with a dilution rate of 1:200 at 4 °C overnight. The endogenous peroxidase was quenched in 0.3% H₂O₂ for 30 minutes and the sections were incubated with biotinylated secondary antibody (IMMPRESS® HRP Anti-Rabbit IgG kit, Vector Laboratory, Catalog number, MP-7401–15) for 30 minutes at room temperature and the stain was developed by incubating with Metal Enhanced DAB Substrate (IMMPACT® DAB kit, Vector Laboratories, SK-4105). Nuclei were counterstained with hematoxylin (Ricca chemical 3536–16). All washes between steps were performed in either double-distilled water or TNT buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.05 % Tween).

In situ sampling and quantification of periostin in mouse periosteum and skin through plasmonic-fluor enhanced microneedle:

Mouse periostin Duoset ELISA kit (R&D systems, catalog number DY2955, lot number P217047) was employed in the study. To sample periostin in mouse periosteum and dermal ISF, microneedle patches were pre-functionalized with periostin capture antibody (PART # 842318) in a 24 well plate clamping on a magnetic plate separator, followed by washing with PBST and blocking with reagent diluent (1X PBS containing 3% BSA, 0.2 μm filtered). Skin above periosteum layer were carefully incised using scissors. A microneedle patch was administered on periosteum and another patch was administered on dorsal skin for 10 minutes, under anesthesia. Followed by another 30 minutes of blocking with reagent diluent (1X PBS containing 3% BSA, 0.2 μm filtered), microneedle patches were exposed to biotinylated detection antibody (PART # 842319), streptavidin-800CW and plasmonic-fluor. Fluorescence maps of microneedle patches were imaged using LICOR CLx fluorescence imager using the same scanning parameters as described above. Blood was collected by cardiac puncture and serum was stored at −20 °C for further analysis. Periostin ELISA for mouse serum were implemented in similar approach on 96 well microtiter plate.

Statistics.

For analyzing the statistical difference between two groups, an unpaired one-tailed t-test with Welch’s correction. For analyzing the statistical difference between each data point in two groups, the two-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test was used. For analyzing the statistical difference between two or more groups, the one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test was used. Statistical significance of the data was calculated at 95% (P<0.05) confidence intervals. All values are expressed as mean ± s.d. GraphPad Prism 8 was used for all statistical analysis. We employed four-parameter logistic or polynomial fit to calculate the LOD in the standard curves of bioassays. The LOD is defined as the analyte concentration corresponding to the mean fluorescence intensity of blank plus three times of its standard deviation (mean+3σ). Origin 2016 was employed for calculating the LOD.