Detection Of Specific Proteins Research Articles

Integrated CRISPR/Cas12a with terminal protection strategy for homogeneous protein assay

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3’-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans-cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans-cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope.

Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.

Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibriumbetween B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Shedding Light on Intracellular Proteins using Flow Cytometry.

Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed.

A Simple Sandwich Electrochemical Immunosensor for Rapid Detection of the Alzheimer's Disease Biomarker Tau Protein.

As a typical biomarker of Alzheimer's disease, rapid and specific detection of tau protein can help improve the early diagnosis and prognosis of the disease. In this study, a simple sandwich electrochemical immunosensor was developed for rapid detection of tau protein. Primary monoclonal antibodies (mAb1) against the middle domain of tau protein (amino acids 189-195) were immobilized on the gold electrode surface through a self-assembled monolayer (SAM) of 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP). Then the tau protein was captured through the specific adsorption between the antigen and the antibody, resulting in a change in the impedance. Secondary monoclonal antibodies (mAb2) against the N-terminal region of tau protein were used for further amplification of the binding reaction between mAb1 and tau protein. A linear correlation between the total change in impedance and the logarithm of tau concentration was found from 2 × 10-6 mg mL-1 to 2 × 10-3 mg mL-1, with a detection limit as low as 1 × 10-6 mg mL-1. No significant interference was observed from human serum albumin. Furthermore, the fabricated sandwich immunosensor successfully detected target tau protein in artificial cerebrospinal fluid (aCSF) samples, indicating good potential for clinical applications in the future.

High performance THz metasurface sensor based on modified-SWCNTs film for femtomolar protein detection

Terahertz (THz) metasurface sensor plays an important role in environmental monitoring, biomedical diagnostics, and materials science. However, most traditional THz metasurfaces for trace biological substance detection either suffer from high ohmic loss, or cannot achieve ultra-low concentration detection at the femtomolar level, which limits the application range of THz metasurfaces. Here, we propose a novel metasurface based on modified single-walled carbon nanotubes (SWCNTs) film for specific detection of SAA protein in femtomolar concentration. By modifying the SWCNTs film with concentrated sulfuric acid and introducing functionalized gold nanoparticles (Au-NPs), not only the non-resonant loss of the SWCNTs film was greatly reduced, but also the specific selection of trace SAA proteins was achieved. We experimentally demonstrated that the sensitivity of this THz metasurface sensor for SAA protein detection is 37.5 GHz/fM. In contrast to conventional metal or dielectric metasurfaces, because the unique mesh-like structure of the SWCNTs film can greatly increase the interaction between trace substances and the sensor, this new modifiable metasurface can achieve the lowest detection limit of 0.1 fM, which has increased by an order of magnitude. Our results provide a new and promising method for THz metasurfaces to realize high-performance biosensors.

Peptide-Based Turn-On Fluorescent Probes for Highly Specific Detection of Survivin Protein in the Cancer Cells

Peptide-Based Turn-On Fluorescent Probes for Highly Specific Detection of Survivin Protein in the Cancer Cells

Investigating the impact of empagliflozin on the retina of diabetic mice.

Diabetic retinopathy (DR) frequently results in compromised visual function, with hyperglycemia-induced disruption of the blood-retinal barrier (BRB) through various pathways as a critical mechanism. Existing DR treatments fail to address early and potentially reversible microvascular alterations. This study examined the effects of empagliflozin (EMPA), a selective Sodium-glucose transporter 2 (SGLT2) inhibitor, on the retina of db/db mice. The objective of this study is to investigate the potential role of EMPA in the prevention and delay of DR. db/db mice were randomly assigned to either the EMPA treatment group (db/db + Emp) or the model group (db/db), while C57 mice served as the normal control group (C57). Mice in the db/db + Emp group received EMPA for eight weeks. Body weight, fasting blood glucose (FBG), and blood VEGF were subsequently measured in all mice, along with the detection of specific inflammatory factors and BRB proteins in the retina. Retinal SGLT2 protein expression was compared using immunohistochemical analysis, and BRB structural changes were observed via electron microscopy. EMPA reduced FBG, blood VEGF, and retinal inflammatory factors TNF-α, IL-6, and VEGF levels in the eye tissues of db/db mice. EMPA also increased Claudin-1, Occludin-1, and ZO-1 levels while decreasing ICAM-1 and Fibronectin, thereby preserving BRB function in db/db mice. Immunohistochemistry revealed that EMPA reduced SGLT2 expression in the retina of diabetic mice, and electron microscopy demonstrated that EMPA diminished tight junction damage between retinal vascular endothelial cells and prevented retinal vascular basement membrane thickening in diabetic mice. EMPA mitigates inflammation and preserves BRB structure and function, suggesting that it may prevent DR or serve as an effective early treatment for DR.

Gold structured nanourchin as anesthetic biosensor for determining neuroprotective effect

Research has proven that confusion, delirium, and cognitive dysfunction are the common symptoms caused by anesthesia. In general, neuroproteins fluctuation is common during the process of anesthetic treatment. Among others, tau protein was found to the higher fluctuations after the anesthetic process, and it is highly associated with various neuro-related diseases. Monitoring the level of tau protein helps to identify the neuro-related problems during and after the anesthetic processes. This research was focused on developing a tau electrochemical impedance biosensor on aptamer and antibody-modified electrodes. Aptamer and antibody were immobilized on the gold nanourchin with a chemical linker. Gold nanourchin enhances the probe of aptamer and antibody attachment on the electrode and improves the tau protein detection as low as 1 fM [y = 1.5604x − 1.5643; R2 = 0.9681], determined on a linear range from 1 fM to 1 nM. Further, control performances with complementary aptamer and non-immune antibodies fail to change the response of the current, indicating the specific detection of tau protein. This tau sensor detects the tau protein in its lower level and helps to identify the neuro-related problem caused by anesthetics.

Multimetal-Based Metal-Organic Framework System for the Sensitive Detection of Heart-Type Fatty Acid Binding Protein in Electrochemiluminescence Immunoassay.

In this work, an electrochemiluminescence (ECL) quenching system using multimetal-organic frameworks (MMOFs) was proposed for the sensitive and specific detection of heart-type fatty acid-binding protein (H-FABP), a marker of acute myocardial infarction (AMI). Bimetallic MOFs containing Ru and Mn as metal centers were synthesized via a one-step hydrothermal method, yielding RuMn MOFs as the ECL emitter. The RuMn MOFs not only possessed the strong ECL performance of Ru(bpy)32+ but also maintained high porosity and original metal active sites characteristic of MOFs. Moreover, under the synergistic effect of MOFs and Ru(bpy)32+, RuMn MOFs have more efficient and stable ECL emission. The trimetal-based MOF (FePtRh MOF) was used as the ECL quencher because of the electron transfer between FePtRh MOFs and RuMn MOFs. In addition, active intramolecular electron transfer from Pt to Fe or Rh atoms also occurred in FePtRh MOFs, which could promote intermolecular electron transfer and improve electron transfer efficiency to enhance the quenching efficiency. The proposed ECL immunosensor demonstrated a wide dynamic range and a low detection limit of 0.01-100 ng mL-1 and 6.8 pg mL-1, respectively, under optimal conditions. The ECL quenching system also presented good specificity, stability, and reproducibility. Therefore, an alternative method for H-FABP detection in clinical diagnosis was provided by this study, highlighting the potential of MMOFs in advancing ECL technology.

Pathogen perception and signaling in plant immunity.

Plant diseases are a constant and serious threat to agriculture and ecological biodiversity. Plants possess a sophisticated innate immunity system capable of detecting and responding to pathogen infection to prevent disease. Our understanding of this system has grown enormously over the past century. Early genetic descriptions of plant disease resistance and pathogen virulence were embodied in the gene-for-gene hypothesis, while physiological studies identified pathogen-derived elicitors that could trigger defense responses in plant cells and tissues. Molecular studies of these phenomena have now coalesced into an integrated model of plant immunity involving cell surface and intracellular detection of specific pathogen-derived molecules and proteins culminating in the induction of various cellular responses. Extracellular and intracellular receptors engage distinct signaling processes but converge on many similar outputs with substantial evidence now for integration of these pathways into interdependent networks controlling disease outcomes. Many of the molecular details of pathogen recognition and signaling processes are now known, providing opportunities for bioengineering to enhance plant protection from disease. Here we provide an overview of the current understanding of the main principles of plant immunity, with an emphasis on the key scientific milestones leading to these insights.

Pseudo-Luciferase Activity of the SARS-CoV-2 Spike Protein for Cypridina Luciferin.

Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

Flow cytometry-based diagnostic approach for inborn errors of immunity: experience from Algeria.

In this study, we retrospectively reviewed the use of flow cytometry (FCM) in the diagnosis of inborn errors of immunity (IEIs) at a single center in Algeria. Sharing insights into our practical experience, we present FCM based diagnostic approaches adapted to different clinical scenarios. Between May 2017 and February 2024, pediatric and adult patients presenting with clinical features suggestive of immunodeficiency were subjected to FCM evaluation, including lymphocyte subset analysis, detection of specific surface or intracellular proteins, and functional analysis of immune cells. Over a nearly seven-year period, our laboratory diagnosed a total of 670 patients (372 (55.5%) males and 298 (44.5%) females), distributed into 70 different IEIs belonging to 9 different categories of the International Union of Immunological Societies classification. FCM was used to diagnose and categorize IEI in 514 patients (76.7%). It provided direct diagnostic insights for IEIs such as severe combined immunodeficiency, Omenn syndrome, MHC class II deficiency, familial hemophagocytic lymphohistiocytosis, and CD55 deficiency. For certain IEIs, including hyper-IgE syndrome, STAT1-gain of function, autoimmune lymphoproliferative syndrome, and activated PI3K delta syndrome, FCM offered suggestive evidence, necessitating subsequent genetic testing for confirmation. Protein expression and functional assays played a crucial role in establishing definitive diagnoses for various disorders. To setup such diagnostic assays at high and reproducible quality, high level of expertise is required; in house reference values need to be determined and the parallel testing of healthy controls is highly recommended. Flow cytometry has emerged as a highly valuable and cost-effective tool for diagnosing and studying most IEIs, particularly in low-income countries where access to genetic testing can be limited. FCM analysis could provide direct diagnostic insights for most common IEIs, offer clues to the underlying genetic defects, and/or aid in narrowing the list of putative genes to be analyzed.

Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1.

The study of host-pathogen interaction often requires interrogating the protein-protein interactions and examining post-translational modifications of the proteins. Traditional protein detection strategies are limited in their sensitivity, specificity, and multiplexing capabilities. The Proximity Ligation Assay (PLA), a versatile and powerful molecular technique, can overcome these limitations. PLA blends the specificity of antibodies, two antibodies detecting two different epitopes on the same or two different proteins, with the amplification efficiency of a polymerase to allow highly specific and sensitive detection of low-abundant proteins, protein-protein interactions, or protein modifications. In this protocol, we describe the application of PLA to detect the proximity between HIV-1 Tat with one of its cellular partners, p65, in an infected host cell. The protocol could be applied to any other context with slight modifications. Of note, PLA can only confirm the physical proximity between two epitopes or proteins; however, the proximity need not necessarily allude to the functional interaction between the two proteins.

Metallic nanostructure-based aptasensors for robust detection of proteins.

There is a significant need for fast, cost-effective, and highly sensitive protein target detection, particularly in the fields of food, environmental monitoring, and healthcare. The integration of high-affinity aptamers with metal-based nanomaterials has played a crucial role in advancing the development of innovative aptasensors tailored for the precise detection of specific proteins. Aptamers offer several advantages over commonly used molecular recognition methods, such as antibodies. Recently, a variety of metal-based aptasensors have been established. These metallic nanomaterials encompass noble metal nanoparticles, metal oxides, metal-carbon nanotubes, carbon quantum dots, graphene-conjugated metallic nanostructures, as well as their nanocomposites, metal-organic frameworks (MOFs), and MXenes. In general, these materials provide enhanced sensitivity through signal amplification and transduction mechanisms. This review primarily focuses on the advancement of aptasensors based on metallic materials for the highly sensitive detection of protein targets, including enzymes and growth factors. Additionally, it sheds light on the challenges encountered in this field and outlines future prospects. We firmly believe that this review will offer a comprehensive overview and fresh insights into metallic nanomaterials-based aptasensors and their capabilities, paving the way for the development of innovative point-of-care (POC) diagnostic devices.

B-083 Novel High Affinity Monoclonal Antibodies for Specific Detection of Neurofilament Light Protein in Serum and Cerebrospinal Fluid

Abstract Background Neurofilaments are very important protein components of axons, and they maintain the function of nervous system. Cerebrospinal fluid (CSF) and serum neurofilament levels elevate due to damages in nervous system making neurofilaments promising biomarkers for neural diseases and injuries, such as Alzheimer’s disease, traumatic brain injury and Multiple sclerosis. Based on their size, neurofilaments are divided in groups: Neurofilament light (NfL), neurofilament medium (NfM), neurofilament heavy (NfH), α-internexin, and peripherin. Of these, NfL is considered to be the most promising biomarker since it is the most abundant form of neurofilaments. Methods We have developed four mouse monoclonal antibodies against human NfL. These antibodies, designated as Anti-h NfL 12601, 12603, 12604 or 12605, were tested as pairs in sandwich fluorescence-based immunoassay (FIA) with NfL spiked serum and buffer to determine linear measuring ranges and the matrix effect. Furthermore, the ability of the antibodies to measure NfL in clinical samples was tested in sandwich FIA by testing 10 CSF samples in the range of 0 to 5 083 pg/mL. The antibodies were also compared against commercially available reference antibodies in FIA. Kinetic parameters were determined with bio-layer interferometry (BLI). Results In sandwich FIA, the widest linear measuring range of 10 to 40 960 pg/mL of recombinant human NfL spiked in normal serum was achieved with antibody pair 12603 and 12604. In contrast, the commercial reference antibody pair achieved a linear measuring range of 2560 to 40 960 pg/mL. Matrix effect caused by serum was negligible. With clinical samples, the best correlation with the reference method (NF-light CSF ELISA from Uman Diagnostics) with a coefficient of correlation of 0.986 was achieved with antibody pair 12604 and 12601. This pair also showed a good correlation with the commercial reference pair with a coefficient of correlation of 0.984. In kinetics measurements, none of four antibodies showed dissociation indicating very high affinity. The fastest association of 4.0 × 105 1/Ms was achieved with antibody 12605. Conclusion These results demonstrate that the newly developed mouse monoclonal NfL antibodies are promising tools for development of diagnostic immunoassays measuring NfL levels in human serum and CSF samples. Comparison against commercial reference antibodies indicates that higher NfL detection sensitivity and wider detection ranges can be achieved with antibody pairs developed in this study. Additionally, the kinetic measurement results forecast these antibodies to be suitable for fast reaction kinetic assay formats, such as lateral flow assays, which might become a key resolution in acute neural injury diagnostics.

Gold-coated tilted fiber Bragg gratings for lead ion sensing.

Surface plasmon resonance sensor based on gold-coated tilted fiber Bragg gratings (SPR-TFBGs) are perfectly suited for fine refractometry. Thanks to the functionalization of the gold layer, they can be used for label-free biosensing. They have been largely used for the specific detection of proteins and cells. In this work, we experimentally demonstrate that they are enough sensitive to detect a very small entity like an environmental pollutant. In this context, we report here a bio-functionalization of the SPR-TFBG with thrombin aptamers for lead ion detection. We used aqueous solutions of lead ions with increasing concentrations from 0.001 ppb to 10 ppb. Based on the affinity bending of Pb2+ ions to the thrombin aptamer, we experimentally demonstrated low detection level of lead ion concentration (0.001 ppb) while the saturation limit is meanly fixed by the physical dimension of the sensor and the binding efficiency.

A polydopamine nanospheres modified fiber optic SPR biosensor for specific detection of C-reactive protein

C-reactive protein (CRP) is a vital inflammatory marker in humans and a key single marker of cardiovascular diseases. In this paper, the concentration of CRP was specifically detected by a polydopamine nanospheres (PDA-NPs) developed surface plasmon resonance (SPR) sensor with high accuracy. PDA-NPs is a self-polymerizing material that can be obtained from dopamine through the mussel adhesion effect. The PDA-NPs (diameter ≈ 100 nm) modified SPR sensor exhibits enhanced refractive index (RI) sensitivity and bio-sensitivity attributed to the large specific surface area and high surface reactivity of the PDA-NPs. The developed sensor demonstrates a RI sensitivity of 2427.68 nm/RIU, presenting a 55.7% increase compared to the raw sensor (RI sensitivity was 1559.08 nm/RIU). Moreover, the developed biosensor is used to detect CRP specifically. The bio-sensitivity reaches 0.09 nm/(µg/mL), while the limit of detection (LOD) reaches 0.22 µg/mL. These findings indicate that the developed biosensor can specifically detect CRP with a low LOD. Therefore, this PDA-NPs modified biosensor has great potential in clinical detection and other bio-detection filed.

Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.