Plasmonically Enhanced Ultrasensitive Epitope-Specific Serologic Assay for COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread and resulted in the global pandemic of COVID-19. Although IgM/IgG serology assay has been widely used, with the entire spike or nucleocapsid antigens, they only indicate the presence or absence of antibodies against these proteins but are not specific to the neutralization antibodies, therefore providing only generic information about infection stage and possible future immune protection. Novel technologies enabling easy-to-use and sensitive detection of multiple specific antibodies simultaneously will facilitate precise diagnosis of infection stage, prediction of clinical outcomes, and evaluation of future immune protection upon viral exposure or vaccination. Here, we demonstrate a rapid and ultrasensitive quantification method for epitope-specific antibodies, including different isotypes and subclasses, in a multiplexed manner. Using an ultrabright fluorescent nanolabel, plasmonic-fluor, this novel assay can be completed in 20 min and more importantly, the limit of detection of the plasmon-enhanced immunoassay for SARS-CoV-2 antibodies is as much as 100-fold lower compared to the assays relying on enzymatic amplification of colorimetric signals. Using convalescent patient plasma, we demonstrate that this biodetection method reveals the patient-to-patient variability in immune response as evidenced by the variations in whole protein and epitope-specific antibodies. This cost-effective, rapid, and ultrasensitive plasmonically enhanced multiplexed epitope-specific serological assay has the potential to be broadly employed in the detection of specific antibodies, which may benefit the advanced epidemiology studies and enable improvement of the clinical outcomes and prediction of the future protection against the SARS-CoV-2.

Ultrabright plasmonic fluor nanolabel-enabled detection of a urinary ER stress biomarker in autosomal dominant tubulointerstitial kidney disease.

Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is the most common nonpolycystic genetic kidney disease, but it remains unrecognized due to its clinical heterogeneity and lack of screening test. Moreover, the fact that the clinical feature is a poor predictor of disease outcome further highlights the need for the development of mechanistic biomarkers in ADTKD. However, low abundant urinary proteins secreted by thick ascending limb cells, where UMOD is synthesized, have posed a challenge for the detection of biomarkers in ADTKD-UMOD. In the CRISPR/Cas9-generated murine model and patients with ADTKD-UMOD, we found that immunoglobulin heavy chain-binding protein (BiP), an endoplasmic reticulum chaperone, was exclusively upregulated by mutant UMOD in the thick ascending limb and easily detected by Western blot analysis in the urine at an early stage of disease. However, even the most sensitive ELISA failed to detect urinary BiP in affected individuals. We therefore developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA) to quantify urinary BiP concentration by harnessing the newly invented ultrabright fluorescent nanoconstruct, termed "plasmonic Fluor." p-FLISA demonstrated that urinary BiP excretion was significantly elevated in patients with ADTKD-UMOD compared with unaffected controls, which may have potential utility in risk stratification, disease activity monitoring, disease progression prediction, and guidance of endoplasmic reticulum-targeted therapies in ADTKD.NEW & NOTEWORTHY Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is an underdiagnosed cause of chronic kidney disease (CKD). Lack of ultrasensitive bioanalytical tools has hindered the discovery of low abundant urinary biomarkers in ADTKD. Here, we developed an ultrasensitive plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA). p-FLISA demonstrated that secreted immunoglobulin heavy chain-binding protein is an early urinary endoplasmic reticulum stress biomarker in ADTKD-UMOD, which will be valuable in monitoring disease progression and the treatment response in ADTKD.

Bioplasmonic paper-based assay for perilipin-2 non-invasively detects renal cancer.

Renal cell carcinoma (RCC) has poor survival prognosis because it is asymptomatic at an early, more curative stage. Recently, urine perilipin-2 (PLIN-2) was demonstrated to be a sensitive and specific biomarker for the noninvasive, early detection of RCC and an indispensable indicator to distinguish cancer from a benign renal mass. However, current Western blot or ELISA PLIN-2 assays are complicated, expensive, time-consuming or insensitive, making them unsuitable for routine analysis in clinical settings. Here we developed a plasmonic biosensor based on the high refractive index sensitivity of gold nanorattles for the rapid detection of PLIN-2 in patient urine. The paper-based plasmonic assay is highly sensitive and has a dynamic range of 50 pg/ml to 5 µg/ml PLIN-2. The assay is not compromised by variations in urine pH or high concentrations of interfering proteins such as albumin and hemoglobin, making it an excellent candidate for routine clinical applications. The urine PLIN-2 assay readily distinguished patients with pathologically proven clear cell carcinomas of various size, stage and grade (55.9 [39.5, 75.8] ng/ml, median [1st and 3rd quartile]) from age-matched controls (0.3 [0.3, 0.5] ng/ml), patients with bladder cancer (0.5 [0.4, 0.6] ng/ml) and patients with diabetic nephropathy (0.6 [0.4, 0.7] ng/ml). Urine PLIN-2 concentrations were roughly proportional to tumor size (Pearson coefficient 0.59). Thus, this cost-effective and label-free method represents a novel approach to conduct a non-invasive population screen or rapid differential diagnosis of imaged renal masses, significantly facilitating the early detection and diagnosis of RCC.

Urinary aquaporin 1 and perilipin 2: Can these novel markers accurately characterize small renal masses and help guide patient management?

OBJECTIVE: To evaluate the role of urine aquaporin 1 and perilipin 2 as biomarkers adjunct to renal mass biopsy in guiding the management of patients with small renal masses. METHODS: Preoperative aquaporin 1 and perilipin 2 levels in 57 patients with small renal masses undergoing partial nephrectomy were analyzed and compared with postoperative tumor histology. An algorithm was created utilizing aquaporin 1 and perilipin 2 in conjunction with renal mass biopsy. Cut-off values were implemented to maximize biomarker sensitivity and specificity. Renal mass biopsy utilization and intervention were then compared with rates in traditional renal mass biopsy algorithms. RESULTS: All clear cell and papillary renal cell carcinomas were correctly identified and assigned to the treatment path. All benign lesions were correctly sorted to a confirmatory renal mass biopsy path. Two chromophobe masses did not have elevated aquaporin 1 and perilipin 2, and would require renal mass biopsy. Compared with protocols that call for all small renal masses to be biopsied, confirmatory renal mass biopsy could have been safely avoided in 74% of patients with elevated aquaporin 1 and perilipin 2. Compared with protocols that do not utilize renal mass biopsy, surgical intervention would have been avoided in 23% of patients with benign masses. CONCLUSIONS: Aquaporin 1 and perilipin 2 possess high sensitivity and specificity for detecting clear cell and papillary renal cell carcinoma. Use of these markers might compliment renal mass biopsy in the characterization of small renal masses.

Environmental Stability of Plasmonic Biosensors Based on Natural versus Artificial Antibody.

Plasmonic biosensors based on the refractive index sensitivity of localized surface plasmon resonance (LSPR) are considered to be highly promising for on-chip and point-of-care biodiagnostics. However, most of the current plasmonic biosensors employ natural antibodies as biorecognition elements, which can easily lose their biorecognition ability upon exposure to environmental stressors (e.g., temperature and humidity). Plasmonic biosensors relying on molecular imprints as recognition elements (artificial antibodies) are hypothesized to be an attractive alternative for applications in resource-limited settings due to their excellent thermal, chemical, and environmental stability. In this work, we provide a comprehensive comparison of the stability of plasmonic biosensors based on natural and artificial antibodies. Although the natural antibody-based plasmonic biosensors exhibit superior sensitivity, their stability (temporal, thermal, and chemical) was found to be vastly inferior to those based on artificial antibodies. Our results convincingly demonstrate that these novel classes of artificial antibody-based plasmonic biosensors are highly attractive for point-of-care and resource-limited conditions where tight control over transport, storage, and handling conditions is not possible.

Single Molecule Force Spectroscopy to Compare Natural versus Artificial Antibody-Antigen Interaction.

Biorecognition is central to various biological processes and finds numerous applications in virtually all areas of chemistry, biology, and medicine. Artificial antibodies, produced by imprinting synthetic polymers, are designed to mimic the biological recognition capability of natural antibodies, while exhibiting superior thermal, chemical, and environmental stability compared to their natural counterparts. The binding affinity of the artificial antibodies to their antigens characterizes the biorecognition ability of these synthetic nanoconstructs and their ability to replace natural recognition elements. However, a quantitative study of the binding affinity of an artificial antibody to an antigen, especially at the molecular level, is still lacking. In this study, using atomic force microscopy-based force spectroscopy, the authors show that the binding affinity of an artificial antibody to an antigen (hemoglobin) is weaker than that of natural antibody. The fine difference in the molecular interactions manifests into a significant difference in the bioanalytical parameters of biosensors based on these recognition elements.

Hot Spot-Localized Artificial Antibodies for Label-Free Plasmonic Biosensing.

The development of biomolecular imprinting over the last decade has raised promising perspectives in replacing natural antibodies with artificial antibodies. A significant number of reports have been dedicated to imprinting of organic and inorganic nanostructures, but very few were performed on nanomaterials with a transduction function. Herein we describe a relatively fast and efficient plasmonic hot spot-localized surface imprinting of gold nanorods using reversible template immobilization and siloxane co-polymerization. The technique enables a fine control of the imprinting process at the nanometer scale and provides a nanobiosensor with high selectivity and reusability. Proof of concept is established by the detection of neutrophil gelatinase-associated lipocalin (NGAL), a biomarker for acute kidney injury, using localized surface plasmon resonance spectroscopy. The work represents a valuable step towards plasmonic nanobiosensors with synthetic antibodies for label-free and cost-efficient diagnostic assays. We expect that this novel class of surface imprinted plasmonic nanomaterials will open up new possibilities in advancing biomedical applications of plasmonic nanostructures.

The specificity of urinary aquaporin 1 and perilipin 2 to screen for renal cell carcinoma.

PURPOSE: Renal cancer is frequently asymptomatic until late stages of the disease and it has a poor prognosis when not discovered early. AQP1 and PLIN2 are recently discovered, sensitive urine biomarkers of clear cell and papillary kidney cancer. We validated these biomarkers in a second cohort of patients and determined the effect of common kidney diseases on specificity. MATERIALS AND METHODS: Urine samples were obtained from 36 patients with clear cell or papillary kidney cancer, 43 controls, 44 patients with documented urinary tract infection, 24 diagnosed with diabetic nephropathy and 18 diagnosed with glomerulonephritis. Urine levels of AQP1 and PLIN2 normalized to urine creatinine were determined by a sensitive, specific Western blot procedure. RESULTS: Compared with controls, urine AQP1 and PLIN2 levels in patients with kidney cancer were 23-fold and fourfold greater, respectively, and they decreased 83% to 84% after tumor excision. There was a linear correlation between urine AQP1 and PLIN2 levels, and tumor size (each p <0.001). Urine AQP1 and PLIN2 levels in patients with kidney cancer were 11 to 23-fold and 17 to 25-fold greater, respectively, than in patients with the common kidney diseases. CONCLUSIONS: The ability of urine AQP1 and PLIN2 to identify patients with kidney cancer compared to controls was validated in a second cohort of patients. Common kidney diseases do not adversely increase urine AQP1 and PLIN2 levels or decrease their specificity to screen for renal cancer.

Biochemical network analysis of protein-protein interactions to follow-up T1 bladder cancer patients.

Bladder cancer (BCa) is a prevalent disease with a high risk of aggressive recurrence in T1-stage patients. Despite the efforts to anticipate recurrence, a reliable method has yet to be developed. In this work, we employed high-resolution mass spectrometry to compare the urinary proteome of T1-stage BCa patients with recurring versus non-recurring disease to uncover actionable clinical information predicting recurrence. All patients were diagnosed with T1-stage bladder cancer between the ages of 51 and 91, and urine samples were collected before medical intervention. Our results suggest that the urinary myeloperoxidase to cubilin ratio could be used as a new tool for predicting recurrence and that dysregulation of the inflammatory and immune systems may be a key driver of disease worsening. Furthermore, we identified neutrophil degranulation and neutrophil extracellular traps (NETs) as key pathways in the progression of T1-stage BCa. We propose that proteomics follow-up of the inflammatory and immune systems may be useful for monitoring the effectiveness of therapy. SIGNIFICANCE: This article describes how proteomics can be used to characterize tumor aggressiveness in patients with the same diagnosis of bladder cancer (BCa). LC-MS/MS in combination with label free quantification (LFQ) were used to explore potential protein and pathway level changes related to the aggressiveness of the disease in 13 and 17 recurring and non-recurring T1 stage BCa patients. We have shown that the MPO/CUBN protein ratio is a candidate for a urine prognosis tool in BCa. Furthermore, we identify dysregulation of inflammation process as a driver for BCa recurrence and progression. Moreover, we propose using proteomics to track the effectiveness of therapy in the inflammatory and immune systems.

Preservation of Proteins in Human Plasma through Metal-Organic Framework Encapsulation.

Traditional cold chain systems of collection, transportation, and storage of biofluid specimens for eventual analysis pose a huge financial and environmental burden. These systems are impractical in pre-hospital and resource-limited settings, where refrigeration and electricity are not reliable or even available. Here, we develop an innovative technology using metal-organic frameworks (MOFs), a novel class of organic-inorganic hybrids with high thermal stability, as encapsulates for preserving the integrity of protein biomarkers in biofluids under ambient or non-refrigerated storage conditions. We encapsulate prostate-specific antigen (PSA) in whole patient plasma using hydrophilic zeolitic imidazolate framework-90 (ZIF-90) for preservation at 40 °C for 4 weeks and eventual on-demand reconstitution for antibody-based assays with recovery above 95% compared to storage at -20 °C. Without ZIF-90 encapsulation, only 10-30% of the PSA immunoactivity remained. Furthermore, we demonstrate encapsulation of multiple cancer biomarker proteins in whole patient plasma using ZIF-8 or ZIF-90 encapsulants for eventual on-demand reconstitution and analysis after 1 week at 40 °C. Overall, MOF encapsulation of patient biofluids is important as climate change may be affecting the stability and increase costs of maintaining biospecimen cold chain custody for the collection, transportation, and storage of biospecimens prior to analysis or for biobanking regardless of any countries' affluence.

Ultrasensitive lateral-flow assays via plasmonically active antibody-conjugated fluorescent nanoparticles.

Lateral-flow assays (LFAs) are rapid and inexpensive, yet they are nearly 1,000-fold less sensitive than laboratory-based tests. Here we show that plasmonically active antibody-conjugated fluorescent gold nanorods can make conventional LFAs ultrasensitive. With sample-to-answer times within 20 min, plasmonically enhanced LFAs read out via a standard benchtop fluorescence scanner attained about 30-fold improvements in dynamic range and in detection limits over 4-h-long gold-standard enzyme-linked immunosorbent assays, and achieved 95% clinical sensitivity and 100% specificity for antibodies in plasma and for antigens in nasopharyngeal swabs from individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Comparable improvements in the assay's performance can also be achieved via an inexpensive portable scanner, as we show for the detection of interleukin-6 in human serum samples and of the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples. Plasmonically enhanced LFAs outperform standard laboratory tests in sensitivity, speed, dynamic range, ease of use and cost, and may provide advantages in point-of-care diagnostics.

Direct or indirect endothelial cell transforming growth factor-ß receptor activation initiates arteriolar hyalinosis.

Tacrolimus (FK-506) controls organ rejection; however, arteriolar hyalinosis is a frequent complication. By use of mice lacking FK-506-binding protein-12 in endothelial cells, Chiasson and co-workers explored the contribution of endothelial cells and the transforming growth factor-ß pathway to define mechanisms of hyalinosis. Absence of this binding protein removed a tonic inhibition to activate the transforming growth factor-ß system, causing arteriolar hyalinosis. However, tacrolimus can have effects on any biologic process involving receptors with a GS domain.

Bioplasmonic paper as a platform for detection of kidney cancer biomarkers.

We demonstrate that a common laboratory filter paper uniformly adsorbed with biofunctionalized plasmonic nanostructures can serve as a highly sensitive transduction platform for rapid detection of trace bioanalytes in physiological fluids. In particular, we demonstrate that bioplasmonic paper enables rapid urinalysis for the detection of kidney cancer biomarkers in artificial urine down to a concentration of 10 ng/mL. Compared to conventional rigid substrates, bioplasmonic paper offers numerous advantages such as high specific surface area (resulting in large dynamic range), excellent wicking properties (naturally microfluidic), mechanical flexibility, compatibility with conventional printing approaches (enabling multiplexed detection and multimarker biochips), and significant cost reduction.

Plasmonically-enhanced competitive assay for ultrasensitive and multiplexed detection of small molecules.

Novel methods that enable facile, ultrasensitive and multiplexed detection of low molecular weight organic compounds such as metabolites, drugs, additives, and organic pollutants are valuable in biomedical research, clinical diagnosis, food safety and environmental monitoring. Here, we demonstrate a simple, rapid, and ultrasensitive method for detection and quantification of small molecules by implementing a competitive immunoassay with an ultrabright fluorescent nanolabel, plasmonic fluor. Plasmonic-fluor is comprised of a polymer-coated gold nanorod and bovine serum albumin conjugated with molecular fluorophores and biotin. The synthesis steps and fluorescence emission of plasmonic-fluor was characterized by UV-vis spectroscopy, transmission electron microscopy, and fluorescence microscopy. Plasmon-enhanced competitive assay can be completed within 20 min and exhibited more than 30-fold lower limit-of-detection for cortisol compared to conventional competitive ELISA. The plasmon-enhanced competitive immunoassay when implemented as partition-free digital assay enabled further improvement in sensitivity. Further, spatially multiplexed plasmon-enhanced competitive assay enabled the simultaneous detection of two analytes (cortisol and fluorescein). This simple, rapid, and ultrasensitive method can be broadly employed for multiplexed detection of various small molecules in research, in-field and clinical settings.

High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

Sensitivity and specificity of urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 for the diagnosis of renal cell carcinoma.

BACKGROUND/AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are urinary biomarkers of diagnostic relevance in a wide variety of acute and chronic kidney diseases. Their diagnostic sensitivity and specificity for kidney cancer are largely unknown and therefore the subject of this investigation. METHODS: A prospective cohort study was performed to evaluate urine biomarkers for clear-cell and papillary subtypes of renal cancer (67 patients undergoing nephrectomy) and 55 control patients undergoing non-kidney surgery. Urinary KIM-1 and NGAL concentrations were determined by sensitive and specific ELISAs. RESULTS: In renal cancer patients, median NGAL excretion was 0.52 (1st to 3rd quartiles: 0.28-0.82) ng/mg urinary creatinine (U(Cr)) before nephrectomy compared to 0.15 (0.04-0.31) ng/mg U(Cr) in controls (p < 0.001), and there was a modest decrease of 30% after nephrectomy (p < 0.008). NGAL was not correlated to tumor size (r = 0.19, p = 0.27) or stage. Before nephrectomy, KIM-1 excretion was 0.68 (0.40-1.12) ng/mg U(Cr) compared to 0.03 (0.01-0.06) in controls (p < 0.001). There was a linear correlation between KIM-1 excretion before nephrectomy and tumor size (Spearman's r = 0.66, p < 0.001), tumor stage, and a 50% decrease in median KIM-1 concentration 1 month following tumor excision (p < 0.01). Biomarker concentration ranges for renal cancer patients and controls overlapped substantially for NGAL but not KIM-1. CONCLUSION: NGAL is not a sensitive or specific urinary biomarker of kidney cancer. Although KIM-1 had diagnostic sensitivity for kidney cancer, it is well known to reflect many types of kidney injuries, thus limiting its specificity as a diagnostic biomarker for renal cancer.

Enhancing the Stability of COVID-19 Serological Assay through Metal-Organic Framework Encapsulation.

Enzyme-linked immunosorbent assay is widely utilized in serologic assays, including COVID-19, for the detection and quantification of antibodies against SARS-CoV-2. However, due to the limited stability of the diagnostic reagents (e.g., antigens serving as biorecognition elements) and biospecimens, temperature-controlled storage and handling conditions are critical. This limitation among others makes biodiagnostics in resource-limited settings, where refrigeration and electricity are inaccessible or unreliable, particularly challenging. In this work, metal-organic framework encapsulation is demonstrated as a simple and effective method to preserve the conformational epitopes of antigens immobilized on microtiter plate under non-refrigerated storage conditions. It is demonstrated that in situ growth of zeolitic imidazolate framework-90 (ZIF-90) renders excellent stability to surface-bound SARS-CoV-2 antigens, thereby maintaining the assay performance under elevated temperature (40 °C) for up to 4 weeks. As a complementary method, the preservation of plasma samples from COVID-19 patients using ZIF-90 encapsulation is also demonstrated. The energy-efficient approach demonstrated here will not only alleviate the financial burden associated with cold-chain transportation, but also improve the disease surveillance in resource-limited settings with more reliable clinical data.

Plasmonically Enhanced CRISPR/Cas13a-Based Bioassay for Amplification-Free Detection of Cancer-Associated RNA.

Novel methods that enable sensitive, accurate and rapid detection of RNA would not only benefit fundamental biological studies but also serve as diagnostic tools for various pathological conditions, including bacterial and viral infections and cancer. Although highly sensitive, existing methods for RNA detection involve long turn-around time and extensive capital equipment. Here, an ultrasensitive and amplification-free RNA quantification method is demonstrated by integrating CRISPR-Cas13a system with an ultrabright fluorescent nanolabel, plasmonic fluor. This plasmonically enhanced CRISPR-powered assay exhibits nearly 1000-fold lower limit-of-detection compared to conventional assay relying on enzymatic reporters. Using a xenograft tumor mouse model, it is demonstrated that this novel bioassay can be used for ultrasensitive and quantitative monitoring of cancer biomarker (lncRNA H19). The novel biodetection approach described here provides a rapid, ultrasensitive, and amplification-free strategy that can be broadly employed for detection of various RNA biomarkers, even in resource-limited settings.

Gold Nanorod Size-Dependent Fluorescence Enhancement for Ultrasensitive Fluoroimmunoassays.

Plasmon-enhanced fluorescence (PEF) is a simple and highly effective approach for improving the signal-to-noise ratio and sensitivity of various fluorescence-based bioanalytical techniques. Here, we show that the fluorescence enhancement efficacy of gold nanorods (AuNRs), which are widely employed for PEF, is highly dependent on their absolute dimensions (i.e., length and diameter). Notably, an increase in the dimensions (length × diameter) of the AuNRs from 46 × 14 to 120 × 38 nm2 while holding the aspect ratio constant leads to nearly 300% improvement in fluorescence enhancement efficiency. Further increase in the AuNR size leads to a decrease of the fluorescence enhancement efficiency. Through finite-difference time-domain (FDTD) simulation, we reveal that the size-dependent fluorescence enhancement efficiency of AuNR stems from the size-dependent electromagnetic field around the plasmonic nanostructures. AuNRs with optimal dimensions resulted in a nearly 120-fold enhancement in the ensemble fluorescence emission from molecular fluorophores bound to the surface. These plasmonic nanostructures with optimal dimensions also resulted in a nearly 30-fold improvement in the limit of detection of human interleukin-6 (IL-6) compared to AuNRs with smaller size, which are routinely employed in PEF.

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

The detection and quantification of protein biomarkers in interstitial fluid is hampered by challenges in its sampling and analysis. Here we report the use of a microneedle patch for fast in vivo sampling and on-needle quantification of target protein biomarkers in interstitial fluid. We used plasmonic fluor-an ultrabright fluorescent label-to improve the limit of detection of various interstitial fluid protein biomarkers by nearly 800-fold compared with 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 minimally invasive evaluation of the efficiency of a cocaine vaccine, for longitudinal monitoring of the levels of inflammatory biomarkers, and for efficient sampling of the calvarial periosteum-a challenging site for biomarker detection-and the 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 interstitial fluid might facilitate point-of-care diagnostics and longitudinal monitoring.