Biosensor Assay Research Articles

Rational construction of porous cobalt nanoparticle integrated nitrogen doped hollow carbon nanostructures for peptide agonist exendin-4 biosensing

In this study, we designed a point-of-care (POC) testing electrochemical biosensor using an integrated biosensing assay based on hollow-like nitrogen-doped carbon nanostructures combined with cobalt nanoparticles (Co@HNCNs, Co3O4@HNCNs, and CoP@HNCNs). These are functionalized with Anti-Exendin-4 Antibodies (Anti-Ex-4-Abs) and Bovine Serum Albumin (BSA) to create sensitive probes (Co@HNCNs/Anti-Ex-4-Abs/BSA, Co3O4@HNCNs/Anti-Ex-4-Abs/BSA, and CoP@HNCNs/Anti-Ex-4-Abs/BSA) for the ultrasensitive detection of exendin-4 (Ex-4), a peptide agonist used in the treatment of type 2 diabetes mellitus (T2DM). Among the cobalt-based carbon nanostructures, the Co3O4@HNCNs/Anti-Ex-4-Abs/BSA nanoprobe demonstrated superior ability to specifically recognize Ex-4. This was indicated by a significant decrease in the chronoamperometric (CA) i-t current response, facilitating low-level detection of Ex-4. The nanoprobe was capable of detecting Ex-4 concentrations ranging from 1.0 to 90.0 pM, with a sensitivity of 0.60 μA/pM and a limit of detection (LOD) of 0.46 pM (S/N = 3). Furthermore, the Co3O4@HNCNs/Anti-Ex-4-Abs/BSA nanoprobes demonstrated the ability to detect nanomolar levels of Ex-4 in blood serum and urine samples, achieving satisfactory recovery rates of 96–104%. The proposed electrostatic interaction chemistry approach establishes a remarkable platform for constructing a peptide agonist biosensor that is effective for detecting Ex-4 in real human serum and urine samples.

Enzyme-free and highly sensitive detection of human epidermal growth factor receptor-2 based on MNAzyme signal amplification in breast cancer.

As a common cancer biomarker, human epidermal growth factor receptor-2 (HER2) is highly expressed in breast cancer. Consequently, developing a simple and accurate HER2 sensing platform is of great significance for early diagnosis and treatment of breast cancer. Herein, we developed a rapid enzyme-free fluorescent assay biosensor based on MNAzyme signal amplification for breast cancer biomarker, HER2. The MNAzyme consists of multiple parts, including complementary DNA (cDNA) and two parts of DNAzyme (partzyme A/B). Initially, cDNA is blocked by combining with the HER2 aptamer to form a double-stranded DNA. When HER2 is present, cDNA is released as a result of the binding between HER2 and its aptamer. Due to the complementary sequences among cDNA and partzyme A/B, the MNAzyme is successfully assembled to cleave the substrate, recovering the fluorescence output. The MNAzyme biosensor exhibited a low detection limit of 0.02 ng mL-1 and excellent selectivity. Furthermore, the proposed biosensor can also change the recognition element by changing the aptamer sequence to detect various biomarkers, holding great potential for cancer diagnosis and other related biomedical applications.

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover TLR signaling signatures, including potential biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades.

Locking-Fluorescence Signals Regulated CRISPR/Cas12a Biosensor Based on Metal-Organic Framework for Sensitive Detection of Salmonella typhimurium.

The efficient, sensitive, and rapid detection of Salmonella typhimurium (S. typhimurium) in food and food products is important to ensure food safety and health. This study developed a fluorescence biosensing assay that integrated recombinase-aided amplification (RAA) and CRISPR/Cas12a with a zeolitic imidazolate framework-8@fluorescein sodium (ZIF-8@FLS) nanocomposite for the sensitive detection of S. typhimurium. In this approach, using RAA as a preamplification module, CRISPR/Cas12a-AChE as a target recognition and dual-enzyme cascade amplification module, and the prepared ZIF-8@FLS with high porosity and rapid pH responsiveness as a fluorescence signal explosive amplification module, the RAA-CRISPR/Cas12a-ZIF-8@FLS biosensor was constructed. Under optimal conditions, it exhibited an excellent linear relationship for S. Typhimurium, with a sensitive detection limit as low as 1.3 × 102 CFU/mL and could complete sample detection within 2 h relying on the RAA and ZIF-8@FLS explosive fluorescence rapid response, demonstrating its significant advantages in specificity, sensitivity, and reliability in food-borne pathogens detection.

Essential tremor with tau pathology features seeds indistinguishable in conformation from Alzheimer's disease and primary age-related tauopathy.

Neurodegenerative tauopathies are characterized by the deposition of distinct fibrillar tau assemblies whose rigid core structures correlate with defined neuropathological phenotypes. Essential tremor (ET) is a progressive neurological disease that, in some cases, is associated with cognitive impairment and tau accumulation. Consequently, we explored the tau assembly conformation in ET patients with tau pathology using cytometry-based tau biosensor assays. These assays quantify tau prion seeding activity present in brain homogenates based on conversion of intracellular tau-fluorescent protein fusions from a soluble to an aggregated state. Prions exhibit seeding barriers, whereby a specific assembly structure cannot serve as a template for a native monomer if the amino acids are not compatible. We recently exploited the tau prion species barrier to define tauopathies by systematically substituting alanine (Ala) in the tau monomer and measuring its incorporation into seeded aggregates within biosensor cells. The Ala scan precisely classified the conformation of tau seeds from diverse tauopathies. We next studied 18 ET patient brains with tau pathology. Only one case had concurrent high amyloid-β plaque pathology consistent with Alzheimer's disease (AD). We detected robust tau seeding activity in 9 (50%) of the patients. This predominantly localized to the temporal pole and temporal cortex. We examined 8 ET cases with the Ala scan and determined that the amino acid requirements for tau monomer incorporation into aggregates seeded from these ET brain homogenates were identical to those of AD and primary age-related tauopathy (PART), and completely distinct from other tauopathies such as corticobasal degeneration, chronic traumatic encephalopathy, and progressive supranuclear palsy. Based on these studies, tau assembly cores in a pathologically confined subset of ET cases with high tau pathology are identical to AD and PART. This could facilitate more precise diagnosis and therapy for ET patients with cognitive impairment.

Ultrasensitive and multiplexed Gastric cancer biomarkers detection with an integrated electrochemical immunosensing platform

Developing immunosensing platforms capable of simultaneously detecting multiple cancer markers is crucial for clinical diagnosis and biomedical research. Here, we introduce a novel dual-mode electrochemical biosensing assay platform capable of detecting two gastric cancer biomarkers: pepsinogen I (PG I) and pepsinogen II (PG II). Methylene blue (MB) and Prussian blue (PB) were used as dual signal sources to label PG I and PG II, respectively. The platform integrates an ARM STM32F411 microcontroller and an AD5941 analog front-end, which not only facilitates cyclic voltammetry (CV) and differential pulse voltammetry (DPV) with efficacy comparable to commercial electrochemical workstations but also offers data collection and synchronous analysis capabilities, allowing simultaneous output of PG I and PGR (PG I/PG II) values. Equipped with an interactive screen for operational control and result display, the immunosensing platform provides linear detection ranges for PG I (5 pg/mL–100 ng/mL) and PG II (50 pg/mL–200 ng/mL), enabling rapid detection within 5 min. It demonstrates excellent sensitivity and selectivity when comparing serum samples from healthy individuals and gastric cancer patients. The dual-marker detection platform significantly enhances early diagnosis and screening of gastric cancer, offering substantial improvements over single-marker assays. Furthermore, this platform shows potential for detecting multiple biomarkers in various diseases, highlighting its utility for biomedical applications.

Detection of Sulfonamide Antibiotics Using an Elastic Hydrogel Microparticles-Based Optical Biosensor.

Sulfonamide antibiotics were the first synthetic antibiotics on the market and still have a broad field of application. Their extensive usage, wrong disposal, and limited degradation technologies in wastewater treatment plants lead to high concentrations in the environment, resulting in a negative impact on ecosystems and an acceleration of antibiotic resistance. Although lab-based analytical methods allow for sulfonamide detection, comprehensive monitoring is hampered by the nonavailability of on-site, inexpensive sensing technologies. In this work, we exploit functionalized elastic hydrogel microparticles and their ability to easily deform upon specific binding with enzyme-coated surfaces to establish the groundwork of a biosensing assay for the fast and straightforward detection of sulfonamide antibiotics. The detection assay is based on sulfamethoxazole-functionalized hydrogel microparticles as sensor probes and the biomimetic interaction of sulfonamide analytes with their natural target enzyme, dihydropteroate synthase (DHPS). DHPS from S. pneumoniae was recombinantly produced by E. coli and covalently coupled on a glass biochip using a reactive maleic anhydride copolymer coating. Monodisperse poly(ethylene glycol) hydrogel microparticles of 50 μm in diameter were synthesized within a microfluidic setup, followed by the oriented coupling of a sulfamethoxazole derivative to the microparticle surface. In proof-of-concept experiments, sulfamethoxazole, as the most used sulfonamide antibiotic in medical applications, was demonstrated to be specifically detectable above a concentration of 10 μM. With its straightforward detection principle, this assay has the potential to be used for point-of-use monitoring of sulfonamide antibiotic contaminants in the environment.

An automated syringe-based PoC RT-LAMP LFB platform for infectious disease detection from saliva

Decentralized Point-of-Care (PoC) diagnostics hold momentous potential for rapid and accessible viral infection disease detection. Presented is a unique design application of an easy-to-use (plug-and-play) platform for viral detection. The platform leverages a simplified multiplex Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Lateral Flow Biosensor (LFB) assay with a lyophilized master mix, eliminating the need for RNA isolation or special reporting equipment. A user-friendly Saliva Measuring Tube (SMT) ensures accurate saliva volume self-collection, and a Syringe-based PoC (SPoC) platform automates sample treatment, reagent mixing, and temperature control using readily available components and consumables. The platform’s performance was evaluated by multiplexed detection of the SARS-CoV-2 N2 target gene and human ACTB gene from saliva samples. The SPoC platform achieved a detection limit of spiked 500 copies/mL for SARS-CoV-2 and consistent internal control readout. The presented PoC system offers a promising initial step for further development toward a decentralized solution for viral infection testing.

Arginine-Modified Hemin Enhances G-Quadruplex DNAzyme Peroxidase Activity for High Sensitivity Detection.

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli (STEC) from Food Matrices Using the CANARY Biosensor Assay.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.

Digital Immunoassay for Rapid Detection of SARS-CoV-2 Infection in a Broad Spectrum of Animals.

The ability of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to infect a wide-range of species raises significant concerns regarding both human-to-animal and animal-to-human transmission. There is an increasing demand for highly sensitive, rapid, and simple diagnostic assays that can detect viral infection across various species. In this study, we developed a biosensor assay that adapted a monoclonal-antibody (mAb)-based blocking ELISA format into an Activate Capture + Digital Counting (AC + DC)-based immunoassay. The assay employs a photonic crystal (PC) biosensor, gold-nanoparticle (AuNP) tags, SARS-CoV-2 nucleocapsid (N) protein, and specific anti-N mAb to detect antibody responses in animals exposed with SARS-CoV-2. We demonstrated a simple 2-step 15-min test that was capable of detecting as low as 12.5 ng of antibody in controlled standard serum samples. Based on an evaluation of 176 cat serum samples with known antibody status, an optimal percentage of inhibition (PI) cut-off value of 0.588 resulted in a diagnostic sensitivity of 98.3% and a diagnostic specificity of 96.5%. The test is highly repeatable with low variation coefficients of 2.04%, 2.73%, and 4.87% across different runs, within a single run, and on a single chip, respectively. The test was further employed to detect antibody responses in multiple animal species as well as investigate dynamics of antibody response in experimentally infected cats. This test platform provides an important tool for rapid field surveillance of SARS-CoV-2 infection across multiple species.

Rapid, Point-of-Care Microwave Lysis and Electrochemical Detection of Clostridioides difficile Directly from Stool Samples.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer’s disease pathology progression

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

IgG and IgM differentiation in a particle-based agglutination assay by control over antigen surface density.

Point-of-care (POC) testing offers fast and on-site diagnostics and can be crucial against many infectious diseases and in screening. One remaining challenge in serological POC testing is the quantification of immunoglobulin G (IgG) and immunoglobulin M (IgM). Quantification of IgG/IgM can be important to evaluate immunity and to discriminate recent infections from past infections and primary infections from secondary infections. POC tests such as lateral flow immunoassays allow IgG and IgM differentiation; however, a remaining limitation is their incapacity to provide quantitative results. In this work, we show how samples containing IgG or IgM can be distinguished in a nanoparticle-based agglutination biosensing assay by tuning the density of antigens on the nanoparticles' surface. We employ direct STochastic Optical Reconstruction Microscopy to quantify the accessible SARS-CoV-2 trimeric spike proteins conjugated to magnetic nanoparticles at a single-particle level and gain insight into the protein distribution provided by the conjugation procedure. Furthermore, we measure the anti-SARS-CoV-2 IgG/IgM induced agglutination using an optomagnetic readout principle. We show that particles with high antigen density have a relatively higher sensitivity toward IgM compared to IgG, whereas low antigen density provides a relatively higher sensitivity to IgG. The finding paves the way for its implementation for other agglutination-based serology tests, allowing for more accurate disease diagnosis.

Gas6-Axl signal promotes indoor VOCs exposure-induced pulmonary fibrosis via pulmonary microvascular endothelial cells–fibroblasts cross-talk

Volatile organic compounds (VOCs) as environmental pollutants were associated with respiratory diseases. Pulmonary fibrosis (PF) was characterized by an increase of extracellular matrix, leading to deterioration of lung function. The adverse effects on lung and the potential mechanism underlying VOCs induced PF had not been elucidated clearly. In this study, the indoor VOCs exposure mouse model along with an ex vivo biosensor assay was established. Based on scRNA-seq analysis, the adverse effects on lung and potential molecular mechanism were studied. Herein, the results showed that VOCs exposure from indoor decoration contributed to decreased lung function and facilitated pulmonary fibrosis in mice. Then, the whole lung cell atlas after VOCs exposure and the heterogeneity of fibroblasts were revealed. We explored the molecular interactions among various pulmonary cells, suggesting that endothelial cells contributed to fibroblasts activation in response to VOCs exposure. Mechanistically, pulmonary microvascular endothelial cells (MPVECs) secreted Gas6 after VOCs-induced PANoptosis phenotype, bound to the Axl in fibroblasts, and then activated fibroblasts. Moreover, Atf3 as the key gene negatively regulated PANoptosis phenotype to ameliorate fibrosis induced by VOCs exposure. These novel findings provided a new perspective about MPVECs could serve as the initiating factor of PF induced by VOCs exposure.

The New Delhi metallo-β-lactamase-1 biosensor rapidly and accurately detected antibiotic plasma concentrations in cefuroxime-treated patients

ObjectivesTherapeutic drug monitoring (TDM) of β-lactam antibiotics in critically ill patients may benefit dose optimisation, thus improving therapeutic outcomes. However, rapidly and accurately detecting these antibiotics in blood remains a challenge. This research group recently developed a thermometric biosensor called the New Delhi metallo-β-lactamase-1 (NDM-1) biosensor, which detects multiple classes of β-lactam antibiotics in spiked plasma samples. MethodsThis study assessed the NDM-1 biosensor's effectiveness in detecting plasma concentrations of β-lactam antibiotics in treated patients. Seven patients receiving cefuroxime were studied. Plasma samples collected pre- and post-antibiotic treatment were analysed using the NDM-1 biosensor and compared with liquid chromatography coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). ResultsThe biosensor detected plasma samples without dilution, and a brief pre-treatment using a polyvinylidene fluoride filter significantly lowered matrix effects, reducing the running time to 5–8 minutes per sample. The assay's linear range for cefuroxime (6.25–200 mg/L) covered target concentrations during the trough phase of pharmacokinetics in critically ill patients. The pharmacokinetic properties of cefuroxime in treated patients determined by the NDM-1 biosensor and the UPLC-MS/MS were comparable, and the cefuroxime plasma concentrations measured by the two methods showed statistically good consistency. ConclusionThese data demonstrate that the NDM-1 biosensor assay is a fast, sensitive, and accurate method for detecting cefuroxime plasma concentrations in treated patients and highlights the NDM-1 biosensor as a promising tool for on-site TDM of β-lactam antibiotics in critically ill patients.

Automating data classification for label-free point-of-care biosensing in real complex samples

Surface-based affinity biosensors present a promising avenue for point-of-care (POC) pathogen detection in real-world samples. While laboratory-based devices commonly employ various techniques to mitigate noise, signal drifts, fluidic artifacts, and other system imperfections, their simple cost-effective POC counterparts designed for field use frequently lack such capabilities. This paper addresses this gap by introducing a procedure for automatically classifying pathogen presence in unprocessed liquids from direct detection data measured by a simple POC quartz crystal microbalance sensor device. The procedure integrates classical analytical tools such as filtering, data selection, baseline de-drifting, and result calculation in tailored successive steps, considering the nature of the sensor signal and the challenges posed by real-world media. We show that the developed procedure exhibits exceptional robustness across different biosensing assays and complex real-world media. Through optimizing parameters using diverse datasets encompassing Escherichia coli O157:H7 (E. coli) and SARS-CoV-2 detection in various media including food-derived matrices and cell culture media, we achieved rates of successful detection as high as 80.8 % and 90.9 % for E. coli and SARS-CoV-2, respectively, without extensive machine learning. Furthermore, we analyse the sensitivity of the procedure to variations of input parameters and with examples discuss key factors influencing overall procedure accuracy. Our results suggest that this exceptionally robust method holds potential as a straightforward tool for automating sample classification in point-of-care diagnostics, underpinning its promising broader applicability.

Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π–π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10−17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

Variable CGRP family peptide signaling durations and the structural determinants thereof

Calcitonin gene-related peptides alpha and beta (αCGRP, βCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, −2, or −3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1–47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8–47) and AM2/IMD-53 exhibited even longer duration signaling than the 1–47 fragment. AM2/IMD(8–47) was a striking 8-fold longer acting than AM(13–52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1–52) and (13–52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. βCGRP was ∼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of βCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.