Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein

Photoaffinity-based target identification has received recent attention as an efficient research tool for chemical biology and drug discovery. The major obstacle of photoaffinity-based target identification is the nonspecific interaction between target identification probes and nontarget proteins. Consequently, the rational design of photoaffinity linkers has been spotlighted for successful target identification. These nonspecific interactions have been considered as random events, and therefore no systematic investigation has been conducted regarding nonspecific interactions between proteins and photoaffinity linkers. Herein, we report the protein-labeling analysis of photoaffinity linkers containing three photoactivatable moieties: benzophenone, diazirine, and arylazide. Each photoaffinity linker binds to a different set of proteins in a structure-dependent manner, in contrast to the previous conception. The list of proteins labeled by each photoaffinity linker was successfully used to eliminate the nonspecific binding proteins from target candidates, thereby increasing the success rate of target identification

Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein

Photoaffinity-based target identification has received recent attention as an efficient research tool for chemical biology and drug discovery. The major obstacle of photoaffinity-based target identification is the nonspecific interaction between target identification probes and nontarget proteins. Consequently, the rational design of photoaffinity linkers has been spotlighted for successful target identification. These nonspecific interactions have been considered as random events, and therefore no systematic investigation has been conducted regarding nonspecific interactions between proteins and photoaffinity linkers. Herein, we report the protein-labeling analysis of photoaffinity linkers containing three photoactivatable moieties: benzophenone, diazirine, and arylazide. Each photoaffinity linker binds to a different set of proteins in a structure-dependent manner, in contrast to the previous conception. The list of proteins labeled by each photoaffinity linker was successfully used to eliminate the nonspecific binding proteins from target candidates, thereby increasing the success rate of target identification

Analyses of Intravesicular Exosomal Proteins Using a Nano-Plasmonic System

Extracellular vesicles (EVs), including exosomes, are nanoscale membrane particles shed from cells and contain cellular proteins whose makeup could inform cancer diagnosis and treatment. Most analyses have focused on surface proteins while analysis of intravesicular proteins has been more challenging. Herein, we report an EV screening assay for both intravesicular and transmembrane proteins using a nanoplasmonic sensor. Termed iNPS (intravesicular nanoplasmonic system), this platform used nanohole-based surface plasmon resonance (SPR) for molecular detection. Specifically, we (i) established a unified assay protocol to detect intravesicular as well as transmembrane proteins; and (ii) engineered plasmonic substrates to enhance detection sensitivity. The resulting iNPS enabled sensitive (0.5 μL sample per marker) and high-throughput (a 10 × 10 array) detection for EV proteins. When applied to monitor EVs from drug-treated cancer cells, the iNPS assay revealed drug-dependent unique EV protein signatures. We envision that iNPS could be a powerful tool for comprehensive molecular screening of EVs

Integrated Magneto–Electrochemical Sensor for Exosome Analysis

Extracellular vesicles, including exosomes, are nanoscale membrane particles that carry molecular information on parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magneto–electrochemical assay: exosomes are immunomagnetically captured from patient samples and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables (i) highly sensitive, cell-specific exosome detection and (ii) sensor miniaturization and scale-up for high-throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the simultaneous profiling of multiple protein markers within an hour, outperforming conventional methods in assay sensitivity and speed

Integrated Magneto-Chemical Sensor For On-Site Food Allergen Detection

Adverse food reactions, including food allergies, food sensitivities, and autoimmune reaction (<i>e</i>.<i>g</i>., celiac disease) affect 5–15% of the population and remain a considerable public health problem requiring stringent food avoidance and epinephrine availability for emergency events. Avoiding problematic foods is practically difficult, given current reliance on prepared foods and out-of-home meals. In response, we developed a portable, point-of-use detection technology, termed integrated exogenous antigen testing (<i>i</i>EAT). The system consists of a disposable antigen extraction device coupled with an electronic keychain reader for rapid sensing and communication. We optimized the prototype <i>i</i>EAT system to detect five major food antigens in peanuts, hazelnuts, wheat, milk, and eggs. Antigen extraction and detection with <i>i</i>EAT requires <10 min and achieves high-detection sensitivities (<i><i>e</i>.<i>g</i>.</i>, 0.1 mg/kg for gluten, lower than regulatory limits of 20 mg/kg). When testing under restaurant conditions, we were able to detect hidden food antigens such as gluten within “gluten-free” food items. The small size and rapid, simple testing of the <i>i</i>EAT system should help not only consumers but also other key stakeholders such as clinicians, food industries, and regulators to enhance food safety

Integrated Biosensor for Rapid and Point-of-Care Sepsis Diagnosis

Sepsis is an often fatal condition that arises when the immune response to an infection causes widespread systemic organ injury. A critical unmet need in combating sepsis is the lack of accurate early biomarkers that produce actionable results in busy clinical settings. Here, we report the development of a point-of-care platform for rapid sepsis detection. Termed IBS (integrated biosensor for sepsis), our approach leverages (i) the pathophysiological role of cytokine interleukin-3 (IL-3) in early sepsis and (ii) a hybrid magneto-electrochemical sensor for IL-3 detection. The developed platform produces test results within 1 h from native blood samples and detects IL-3 at a sensitivity of <10 pg/mL; this performance is >5-times faster and >10-times more sensitive than conventional enzyme-linked immunoadsorbent assays, the current gold standard. Using clinical samples, we show that elevated plasma IL-3 levels are associated with high organ failure rate and thus greater risk of mortality, confirming the potential of IL-3 as a sepsis diagnostic biomarker. With further system development (<i>e</i>.<i>g</i>., full automation, data security measures) and rigorous validation studies, the compact and fast IBS could be a practical clinical tool for timely diagnosis and proactive treatment of sepsis

Integrated Biosensor for Rapid and Point-of-Care Sepsis Diagnosis

Sepsis is an often fatal condition that arises when the immune response to an infection causes widespread systemic organ injury. A critical unmet need in combating sepsis is the lack of accurate early biomarkers that produce actionable results in busy clinical settings. Here, we report the development of a point-of-care platform for rapid sepsis detection. Termed IBS (integrated biosensor for sepsis), our approach leverages (i) the pathophysiological role of cytokine interleukin-3 (IL-3) in early sepsis and (ii) a hybrid magneto-electrochemical sensor for IL-3 detection. The developed platform produces test results within 1 h from native blood samples and detects IL-3 at a sensitivity of <10 pg/mL; this performance is >5-times faster and >10-times more sensitive than conventional enzyme-linked immunoadsorbent assays, the current gold standard. Using clinical samples, we show that elevated plasma IL-3 levels are associated with high organ failure rate and thus greater risk of mortality, confirming the potential of IL-3 as a sepsis diagnostic biomarker. With further system development (<i>e</i>.<i>g</i>., full automation, data security measures) and rigorous validation studies, the compact and fast IBS could be a practical clinical tool for timely diagnosis and proactive treatment of sepsis

Integrated Kidney Exosome Analysis for the Detection of Kidney Transplant Rejection

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting