Nonspecific Signal Research Articles

A highly sensitive and specific fluorescent strategy for the detection of Visfatin based on nonlinear hybridization chain reaction

This study established a nonlinear hybridization chain reaction system in which trigger DNA initiates self-sustained assembly of double-stranded hairpin substrates labelled with Sulfo-Cyanine3 (Cy-3) into fluorescent dendritic nanostructures for visfatin detection. The trigger DNA chain initially bound with the anti-visfatin aptamer will be freed with visfatin, which induces continuous chain branching growth and results in an exponential increase in fluorescence signals. Under optimal conditions, the fluorescence intensity increased linearly with visfatin concentrations ranging from 1 ng mL−1 to 100 ng mL−1 (the limit of detection dropped to 0.028 ng mL−1). Furthermore, our study showed that magnetic bead separation minimizes the nonspecific signal of the method. The specificity, stability, and sensitivity of this method were also tested in the study. The results revealed excellent specificity, stability and sensitivity. In the aspect of application evaluation, we tested visfatin in clinical serum samples and obtained credible and accordant results so we suppose that our detection method might have bright prospects in biological molecule technology and early clinical discovery of type 2 diabetes (T2DM).

Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms.

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Self-Immobilizing Quinone Methides for the Fluorescent Sensing of Enzyme Activity

Gaining insight into biological processes relies on sensitive analytical techniques. These often require labeling of biomolecules that help visualize them. Selective covalent labeling without preliminary modification of the biomolecules is an advantageous method. For example, this can be achieved by using probes that are capable of in situ quinone methide (QM) formation. The QM can be masked to give a stable precursor, and the highly reactive form is only generated upon activation by a specific trigger. The in situ formed QM then binds covalently to the nucleophilic side chains of either the target protein or a protein in close proximity. Using fluorogenic probes further improves this method by reducing non-specific background signals, thus improving signal-to-noise ratios. In this review we summarize the development of quinone methide-based probes from mechanism-based inactivation to red-emitting, fluorogenic activity probes, focusing on enzyme-triggered activation.

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3′-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

Development and Characterization of Fluorescent Probes for the G Protein-Coupled Receptor 35.

The orphan G protein-coupled receptor 35 (GPR35) is a potential target for the treatment of pain, inflammation, and metabolic diseases. Although many GPR35 agonists have been discovered, research on functional GPR35 ligands, such as fluorescent probes, is still limited. Herein, we developed a series of GPR35 fluorescent probes by conjugating a BODIPY fluorophore to DQDA, a known GPR35 agonist. All probes exhibited excellent GPR35 agonistic activity and desired spectroscopic properties, as determined by the DMR assay, bioluminescence resonance energy transfer (BRET)-based saturation, and kinetic binding experiments. Notably, compound 15 showed the highest binding potency and the weakest nonspecific BRET binding signal (K d = 3.9 nM). A BRET-based competition binding assay with 15 was also established and used to determine the binding constants and kinetics of unlabeled GPR35 ligands.

Comparison of Near-Infrared Imaging Agents Targeting the PTPmu Tumor Biomarker.

Maximal, safe resection of solid tumors is considered a critical first step in successful cancer treatment. The advent of fluorescence image-guided surgery (FIGS) using non-specific agents has improved patient outcomes, particularly in the case of glioblastoma. Molecularly targeted agents that recognize specific tumor biomarkers have the potential to augment these gains. Identification of the optimal combination of targeting moiety and fluorophore is needed prior to initiating clinical trials. A 20-amino acid peptide (SBK2) recognizing the receptor protein-tyrosine phosphatase mu (PTPmu)-derived tumor-specific biomarker, with or without a linker, was conjugated to three different near-infrared fluorophores: indocyanine green (ICG), IRDye® 800CW, and Tide Fluor™ 8WS. The in vivo specificity, time course, and biodistribution were evaluated for each using mice with heterotopic human glioma tumors that express the PTPmu biomarker to identify component combinations with optimal properties for FIGS. SBK2 conjugated to ICG demonstrated excellent specificity for gliomas in heterotopic tumors. SBK2-ICG showed significantly higher in vivo tumor labeling compared to the Scram-ICG control from 10min to 24h, p < 0.01 at all timepoints, following injection, as well as a significantly higher ex vivo tumor signal at 24h, p < 0.001. Inserting a six-amino acid linker between the targeting peptide and ICG increased the clearance rate and resulted in significantly higher in vivo tumor signal relative to its linker-containing Scrambled control from 10min to 8h, p < 0.05 at all timepoints, after dosing. Agents made with the more hydrophilic IRDye® 800CW and Tide Fluor™ 8WS showed no specific tumor labeling relative to the controls. The IRDye 800CW-conjugated agents cleared within 1h, while the non-specific fluorescent tumor signal generated by the Tide Fluor 8WS-conjugated agents persists beyond 24h. The SBK2 PTPmu-targeting peptide conjugated to ICG specifically labels heterotopic human gliomas grown in mice between 10min and 24h following injection. Similar molecules constructed with more hydrophilic dyes demonstrated no specificity. These studies present a promising candidate for use in FIGS of PTPmu biomarker-expressing tumors.

Surface acoustic wave-based lab-on-a-chip for the fast detection of Legionella pneumophila in water

Surface acoustic wave (SAW) -based immuno-biosensors are used for several applications, thanks to their versatility and faster response than conventional analytical methods. SAW immuno-biosensors can be usefully applied to promptly detect bacteria and prevent bacterial infections that can lead to severe diseases. Here, we present a SAW immuno-biosensor to detect Legionella pneumophila in water. Our device, working at ultra-high frequency (740 MHz), is functionalized with an anti-L. pneumophila antibody to maximize the specificity. We report the characteristic curve of the sensor, calculated measuring bacterial samples at known densities, and its related parameters. We also measure L. pneumophila samples contaminated with different Gram-positive and Gram-negative bacterial species (Escherichia coli and Enterococcus faecium) and samples diluted in mains waters. The proposed device is able to detect L. pneumophila in the range from 1·106 to 1·108 CFU/mL, with a limit of blank of 1.22·106 CFU/mL and a limit of detection of 2.01·106 CFU/mL. The nonspecific signal due to contaminant bacteria is very limited and measurements of L. pneumophila are not affected by contamination. We obtain a good detection also in mains water, representing a realistic matrix for L. pneumophila. Our results are encouraging and pave the way to the use of fast, easy-to-use, reliable and precise sensors to prevent bacterial infections in human activities.

Specific recognition and sensitive quantification of mRNA splice variants via one-pot ligation-dependent loop-mediated isothermal amplification.

Specific recognition and sensitive quantification of mRNA alternative splice variants have been a necessity for exploring the regulatory mechanism of RNA splicing and revealing the association between pre-mRNA splicing and transcriptome function, as well as disease diagnosis. However, their wide abundance range and high sequence homology pose enormous challenges for high sensitivity and selectivity quantification of splice variants. Herein, taking advantage of the excellent specificity of ligation and the powerful nucleic acid replication feature of loop-mediated isothermal amplification (LAMP), we developed a one-pot method (termed one-pot ligation-LAMP) for specific recognition and sensitive quantification of mRNA splicing variants based on two splicing junction-specific stem-loop DNA probe ligation and the subsequently initiating LAMP. The one-pot ligation-LAMP can specifically detect as low as 100 aM mRNA splice variants without any nonspecific signals and quantify them with a wide dynamics range spanning at least six orders of magnitude. We have demonstrated that the one-pot ligation-LAMP is a versatile and practical strategy for accurately quantifying different splicing variants in complex biological samples with high sensitivity all in one tube within 90 min, thereby providing an attractive tool for mRNA splice variant-related studies.

Chemical signal regulated injectable coacervate hydrogels.

In the quest for stimuli-responsive materials with specific, controllable functions, coacervate hydrogels have become a promising candidate, featuring sensitive responsiveness to environmental signals enabling control over sol-gel transitions. However, conventional coacervation-based materials are regulated by relatively non-specific signals, such as temperature, pH or salt concentration, which limits their possible applications. In this work, we constructed a coacervate hydrogel with a Michael addition-based chemical reaction network (CRN) as a platform, where the state of coacervate materials can be easily tuned by specific chemical signals. We designed a pyridine-based ABA triblock copolymer, whose quaternization can be regulated by an allyl acetate electrophile and an amine nucleophile, leading to gel construction and collapse in the presence of polyanions. Our coacervate gels showed not only highly tunable stiffness and gelation times, but excellent self-healing ability and injectability with different sized needles, and accelerated degradation resulting from chemical signal-induced coacervation disruption. This work is expected to be a first step in the realization of a new class of signal-responsive injectable materials.

Development of reverse-transcription loop-mediated isothermal amplification assays for point-of-care testing of human influenza virus subtypes H1N1 and H3N2.

Influenza A virus (IAV) is the most widespread pathogen causing human respiratory infections. Although polymerase chain reaction (PCR)-based methods are currently the mostcommonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In thisstudy, we aimed to develop a more rapid and sensitive method than PCR-based tools todetect IAV using loop-mediated isothermal amplification (LAMP) technology. We designedreverse-transcriptional (RT)-LAMP primers targeting the hemagglutinin gene. RNAs fromreference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers.We optimized the reaction conditions and developed universal reaction conditions for bothLAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMPassays. There was no non-specific signal to 19 non-IAV respiratory viruses, such as influenza B virus, coronaviruses, and respiratory syncytial viruses. Regarding the reaction time, apositive signal was detected within 25 min after starting the reaction. In conclusion, ourRT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 subtypes, making it suitable for point-of-care IAV testing.

Neem leaf glycoprotein mediated epigenetic modification in oral squamous cell carcinoma

BackgroundRecent studies have found out that the initiation and progression of cancer, traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic alterations. Studies also emphasize development of drugs targeting histone deacetylases (HDACs) and DNA methyltransferase inhibitors as an emerging anticancer strategy. Oral squamous cell carcinoma (OSCC) is a deadly disease that comprises 60% of all head and neck squamous cell cancers. The leaves of the Neem tree (Azadirachtaindica) have been used in traditional Ayurvedic medicine for centuries to treat numerous oral maladies and are known to have significant anti-inflammatory properties. NLGP (a glycoprotein isolated from neem leaf) is famous for its potent anti-inflammatory, immunostimulatory and anti-cancer properties. Thus, in the current research proposal we investigated the NLGP mediated various epigenetic modifications in Oral squamous cell carcinoma (OSCC). PurposeThe aim of this work was to clarify if the epigenetic changes in oral squamous cell carcinoma could be mediated by Neem Leaf Glycoprotein (NLGP), which has already been demonstrated as a potent anti-cancer drug. The study's objectives were further expanded to include investigating the impact of NLGP-mediated epigenetic alterations on the Wnt and PI3K pathways. Study DesignIn this research work, we would like to show the epigenetic modification mediated by NLGP on OSCC cell line, SSC-9. We would also like to investigate the involvement of NLGP in certain signal transduction pathway(s) which may play a crucial role in epigenetic modification of oral squamous cell carcinoma. MethodologyChIP assays were performed with MNase-digested chromatin isolated from the NLGP treated and non-treated SCC-9 cells after fixing protein-DNA interactions with 1% formaldehyde. ChIP-grade antibodies and their isotype-Ig antibodies were used to pull-down DNA:Protein complexes. ChIPed samples were used for quantitative polymerase chain reaction (qPCR) analysis and the derived Ct values converted to absolute copy numbers using cloned-DNA plasmid standard dilution curve. Nonspecific signals obtained with IgG-ChIP were subtracted from test samples. Whole cell extracts were prepared from NLGP-treated and non-treated SSC9 (Oral Cancer Squamous) cells with the help of Cell Lysis Buffer (Sigma). Using differentially treated whole cell extracts coimmunoprecipitations were performed with the Universal Magnetic Co-immunoprecipitation kit (Active Motif), as per the manufacturer's specifications. Single blot was sequentially reprobed with multiple antibodies. For each co-immunoprecipitation experiment, isotype IgG antibody served as a negative control and immunoblotting the corresponding whole cell extracts with the primary antibody served as a positive control (input), and most images presented are from the same experiment and same gel exposure. ResultTreatment with NLGP increased percentage enrichment of Histone H3 trimethylation (H3K4me3) and acetylation (H3K14Ac) markers and decreased percentage enrichment of Histone H3 demethylation (H3K9me3, JMJD3) and deacetylation (HDAC1) markers respectively on DNA repair gene loci (ATM, ATR, DDB2), tumour suppressor gene (TP53), Signal transducer and activator of transcription 1 (STAT1) and DNA-dependent RNA polymerase (RNApol2). On further investigation of the molecular pathways that may be involved, we found that NLGP treatment blocks the activation of the DNA PKCs, a component of the Akt pathway and its subsequent phosphorylation of H2AFX and it also inhibits ß-catenin, an integral molecule of the Wnt pathway via increased percentage enrichment of the tri-methylation (H3K4Me3) and acetylation (H3K14Ac) markers thus inhibiting tumorigenesis and cancer progression in OSCC. ConclusionNeem Leaf Glycoprotein has been successfully involved in immuno-regulating the various aspects of the immune system in order to mount an effective anti-cancer action and thus has come forward as an emerging cancer-therapeutic strategy based on natural products. This study also contributes to a better understanding of NLGP's epigenetic modulatory function and paves the path for epigenetic-based therapy for OSCC.

A Proof-of-Concept Electrochemical Skin Sensor for Simultaneous Measurement of Glial Fibrillary Acidic Protein (GFAP) and Interleukin-6 (IL-6) for Management of Traumatic Brain Injuries

This work demonstrates the use of a noninvasive, sweat-based dual biomarker electrochemical sensor for continuous, prognostic monitoring of a Traumatic Brain Injury (TBI) with the aim of enhancing patient outcomes and reducing the time to treatment after injury. A multiplexed SWEATSENSER was used for noninvasive continuous monitoring of glial fibrillary acidic protein (GFAP) and Interleukin-6 (IL-6) in a human sweat analog and in human sweat. Electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) were used to measure the sensor response. The assay chemistry was characterized using Fourier Transform Infrared Spectroscopy (FTIR). The SWEATSENSER was able to detect GFAP and IL-6 in sweat over a dynamic range of 3 log orders for GFAP and 2 log orders for IL-6. The limit of detection (LOD) for GFAP detection in the sweat analog was estimated to be 14 pg/mL using EIS and the LOD for IL-6 was estimated to be 10 pg/mL using EIS. An interference study was performed where the specific signal was significantly higher than the non-specific signal. Finally, the SWEATSENSER was able to distinguish between GFAP and IL-6 in simulated conditions of a TBI in human sweat. This work demonstrates the first proof-of-feasibility of a multiplexed TBI marker combined with cytokine and inflammatory marker detection in passively expressed sweat in a wearable form-factor that can be utilized toward better management of TBIs. This is the first step toward demonstrating a noninvasive enabling technology that can enable baseline tracking of an inflammatory response.

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Spatially resolved in vivo imaging of inflammation-associated mRNA via enzymatic fluorescence amplification in a molecular beacon.

The in vivo optical imaging of RNA biomarkers of inflammation is hindered by low signal-to-background ratios, owing to non-specific signal amplification in healthy tissues. Here we report the design and in vivo applicability, for the imaging of inflammation-associated messenger RNAs (mRNAs), of a molecular beacon bearing apurinic/apyrimidinic sites, whose amplification of fluorescence is triggered by human apurinic/apyrimidinic endonuclease 1 on translocation from the nucleus into the cytoplasm specifically in inflammatory cells. We assessed the sensitivity and tissue specificity of an engineered molecular beacon targeting interleukin-6 (IL-6) mRNA in live mice, by detecting acute inflammation in their paws and drug-induced inflammation in their livers. This enzymatic-amplification strategy may enable the specific and sensitive imaging of other disease-relevant RNAs in vivo.

Polyaniline‐Based Nanostructure Interface for Signal‐to‐Noise Ratio Enhancement in Potentiometric Enzyme‐Free Biosensors

AbstractReduction of nonspecific signals (noise) in electrochemical biosensors is important for the realization of enzyme‐free, point‐of‐care (POC) diagnostic devices. In this study, a simple biosensor interface that simultaneously realizes analyte signal enhancement and noise reduction to increase signal‐to‐noise ratio in a field‐effect transistor‐based potentiometric biosensor is proposed. As a proof‐of‐concept, a phytic‐acid‐doped self‐functionalized polyaniline (pAni)‐based nanostructure is designed. It is shown that the incorporation of gold nanoparticles in the nanostructure increases the signal intensity in serotonin sensing. Moreover, the phenylboronic acid moiety in the pAni backbone and porous nanostructure of the pAni nanostructure reduces the interfering noise generated by catecholamines and human serum albumin. It is believed that this study will contribute to the realization of versatile and multiplexed POC diagnostic devices in the future.

Selective blockade of rat brain T-type calcium channels provides insights on neurophysiological basis of arousal dependent resting state functional magnetic resonance imaging signals.

A number of studies point to slow (0.1–2 Hz) brain rhythms as the basis for the resting-state functional magnetic resonance imaging (rsfMRI) signal. Slow waves exist in the absence of stimulation, propagate across the cortex, and are strongly modulated by vigilance similar to large portions of the rsfMRI signal. However, it is not clear if slow rhythms serve as the basis of all neural activity reflected in rsfMRI signals, or just the vigilance-dependent components. The rsfMRI data exhibit quasi-periodic patterns (QPPs) that appear to increase in strength with decreasing vigilance and propagate across the brain similar to slow rhythms. These QPPs can complicate the estimation of functional connectivity (FC) via rsfMRI, either by existing as unmodeled signal or by inducing additional wide-spread correlation between voxel-time courses of functionally connected brain regions. In this study, we examined the relationship between cortical slow rhythms and the rsfMRI signal, using a well-established pharmacological model of slow wave suppression. Suppression of cortical slow rhythms led to significant reduction in the amplitude of QPPs but increased rsfMRI measures of intrinsic FC in rats. The results suggest that cortical slow rhythms serve as the basis of only the vigilance-dependent components (e.g., QPPs) of rsfMRI signals. Further attenuation of these non-specific signals enhances delineation of brain functional networks.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection.

We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA). High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results. In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA. The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

Design of a Fluorogenic Probe Based on Intramolecular Condensation for Specific Detection of HDAC3.

It is crucial to develop fluorogenic probes for selective targeting of HDACs to explore the roles of HDACs in the tumor onset and progression as well as HDAC-related drug development. However, considerable non-specific signals were produced by spontaneous hydrolysis and undesirable intermolecular attack of the unstable caging moiety in the detection of HDACs with previous probes. To improve the detection specificity, we proposed an intramolecular condensation strategy by the replacement of the traditional acetamide moiety with a trans-enamide unit. Upon deacetylation by HDACs, rapid intramolecular condensation reaction between newly formed terminal aldehyde and hydrazine moiety would occur to afford highly fluorescent hydrazone product. Systematic studies demonstrated that the probe exhibited an extraordinary selectivity for HDAC3 over other HDAC isoforms and interfering substances. The stability and specificity of the indicator make it a powerful tool for HDAC3 activity detection and HDAC3-related drug development.

Highly selective titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles for the enrichment of intact phosphoproteins.

Phosphorylation is one of the most important protein post-translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non-specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β-casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non-fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.