Sensitive Labeling Research Articles

Spectroscopic Stability Studies of Pressure Sensitive Labels Facestock Made from Recycled Post-Consumer Waste and Agro-Industrial By-Products

To support circular economy and sustainability, the use of synthetic polymers should be minimized due to their excessive accumulation in the environment and low biodegradation rate which leads to a global waste problem. Thus, the quota of natural resources should be increased, as well as the use of agriculture and industrial waste or by-products as raw materials in industrial processes. Agro-industrial wastes were found to have high cellulose fiber potential which makes them an excellent resource for paper production. This study investigates the influence of artificial aging test on the stability of commercially available fiber based self-adhesive pressure sensitive labels (PSL), in which the facestock is made of 15% agro-industrial byproducts, 40% post-consumer recycled paper and 45% virgin wood pulp and compared to ones made of biobased polyethylene. The results showed that optical brighteners are present in the composition of fiber based PSL. Moreover, the fluorescence and UV-Visible spectroscopy results indicated that by the action of UV irradiation and temperature. The oxidation of cellulose was not found by FTIR spectroscopy, indicating its high stability.

Carboxylic submetabolome-driven signature characterization of COVID-19 asymptomatic infection

Asymptomatic infection of COVID-19 is a global threat for public health. Unfortunately, the study about metabolic dysregulation of asymptomatic infection is barely investigated. Here, we performed carboxylic submetabolome profiling of serum from 62 asymptomatic and 122 control individuals, by a highly sensitive chemical isotope labelling method. Twenty-one discriminative carboxylic features, including 12-hydroxyeicosatetraenoic acid, cholic acid, glycoursodeoxycholic acid and 15,16-dihydroxyoctadeca-9,12-dienoic acid were discovered to be dysregulated in asymptomatic patients. This panel containing 21 carboxylic features could accurately identify asymptomatic patients based on a random forest model, providing an accuracy of 85.7% with only 3.6% false positive rate and 7.1% false negative rate. The dysregulated metabolites found in asymptomatic patients covered several important pathways, such as arachidonic acid metabolism, synthesis of bile acid, β-oxidation of fatty acids, activation of macrophage and platelet aggregation. This work provided valuable knowledge about serum biomarkers and molecular clues associated with asymptomatic COVID-19 patients.

Label free detection of auramine O by G-quadruplex-based fluorescent turn-on strategy

Auramine o (AO) is a synthetic dye used in paper and textile industries. Although it has been an unauthorized food additive in many countries due to its toxic and carcinogenic possibility, its illegal uses have been detected in certain food products such as pasta, semolina and spices and also in pharmaceuticals. The presence of AO in food products should be monitored, therefore, to minimize the negative health effects on consumers. In this study, a simple, highly sensitive and selective label free detection method was investigated for AO by G-quadruplex-based fluorescent turn-on strategy. The optimum fluorescent detection assay was achieved with a specific G-quadruplex DNA sequence, c-myc, at 400 nM in Tris-HCl buffer at pH 7.4. The linearity of fluorescence intensity depending on AO concentration ranged from 0 to 0.07 µM and LOD and LOQ were 3 nM and 10 nM, respectively. The G-quadruplex-based detection assay was highly specific for AO as compared to other two synthetic food colorings and successfully applied to determine AO in pasta, bulgur and curry powder with recoveries in the range from 70.33% to 106.49%. This G-quadruplex-based label free detection assay has a significant potential to be used in the detection of AO in food products.

In vivoimaging and tracking of exosomes for theranostics

Exosomes are lipid bilayer vesicles released by cells and serve as natural carriers for cell–cell communication. Exosomes provide a promising approach to the diagnosis and treatment of diseases and are considered as an alternative to cell therapy. However, one main restriction in their clinical application is that the current understanding of these vesicles, especially their in vivo behaviors and distributions, remains inadequate. Here, we reviewed the current and emerging methods for in vivo imaging and tracking of exosomes, including fluorescence imaging, bioluminescence imaging, nuclear imaging, X-ray imaging, magnetic resonance imaging, photoacoustic imaging, and multimodal imaging. In vivo imaging and tracking of exosomes by these methods can help researchers further understand their uptake mechanism, biodistribution, migration, function, and therapeutic performance. The pioneering studies in this field can elucidate many unknown exosomal behaviors at different levels. We discussed the advantages and limitations of each labeling and imaging strategy. The advances in labeling and in vivo imaging will expand our understanding of exosomes and promote their clinical application. We finally provide a perspective and discuss several important issues that need to be explored in future research. This review highlights the values of efficient, sensitive, and biocompatible exosome labeling and imaging techniques in disease theranostics.

Identification and Total Synthesis of an Unstable Anticancer Macrolide Presaccharothriolide Z Produced by Saccharothrix sp. A1506.

Saccharothriolides A-F are 10-membered microbial macrolides proposed to be generated from their precursors presaccharothriolides X-Z. Previously, we isolated presaccharothriolide X, and its unique natural prodrug-like properties have intrigued us. However, the other congeners were not detected. Herein, we detected presaccharothriolide Z using our highly sensitive labeling reagent. Moreover, chemical synthesis of presaccharothriolide Z, the first total synthesis of saccharothriolide-class macrolides, was achieved, and the structure and biological activity of presaccharothriolide Z were determined.

Study on the pharmacokinetics of mulberry fruit polysaccharides through fluorescence labeling

A sensitive and efficient fluorescence labeling method was developed and validated for the microanalysis and detection of polysaccharides. Fluorescein isothiocyanate (FITC) was successfully labeled on mulberry fruit polysaccharides (MFP) through a reductive amination reaction with the assistant of tyramine. The fluorescent labeled polysaccharides (FMFP) was identified by fluorescence, UV–visible, flourier transform infrared (FT-IR) and 1H NMR spectrum. Results demonstrated that the labeling efficiency of FMFP was 0.32%, and the FMFP was stable in simulated digestion fluid without cytotoxicity. The pharmacokinetics and biodistribution after administration were analyzed in rats, which indicated that the FMFP obtained could be absorbed in a short time (tmax 0.50 h) but eliminated slowly (t1/2 8.77 ± 1.38 h). At 24 h after administration, the polysaccharide could be tested mainly in intestine, stomach, liver and kidney. The FITC labeling method lays a foundation for investigating the absorption and metabolism of MFP, and provides references for the microanalysis research of bioactive polysaccharides.

5-Hydroxymethylcytosine of Circulating Cell-Free DNA in Plasma: A Novel Non-Invasive Marker for Progression and Prognosis in Multiple Myeloma

Background: Multiple myeloma (MM) universally features profound disruption of their epigenomes with gene-specific DNA hypermethylation linked with clinically aggressive subtypes and prognosis. To date, DNA methylation (i.e., 5-methycytosine) is the most extensively studied cytosine modification. No study has characterized progression and prognostic significance of 5-hydroxymethylcytosine (5hmC), a mark of active demethylation and gene activation, nor did study evaluate 5hmC in cell-free DNA (cfDNA) as a non-invasive “liquid biopsy” mainly due to technological constraints.Methods: In a proof-of-concept study, we used the nano-hmC-Seal, a highly sensitive chemical labeling technique, and the next-generation sequencing to profile 5hmC in plasma cfDNA samples from 19 newly diagnosed, treatment-naïve European American patients with MM (n=9) and its premalignant precursor conditions - monoclonal gammopathy of undetermined significance (MGUS, n=5) and smoldering multiple myeloma (SMM; n=5) from the University of Chicago between 2011-12. Three of the 9 MM patients developed refractory disease or relapsed within 12 months after diagnosis. We profiled 5hmC with 1-2 ng of DNA extracted from 1ml of plasma for library construction. We obtained ~25 million reads per sample, providing a depth of coverage ~600X in terms of gene bodies. We normalized raw fragment counts using DESeq2.Results: We found that in cfDNA, 5hmC loci were enriched within gene bodies, while depleted in CpG islands. Our analysis, based on cis-regulatory elements from the ENCODE (Encyclopedia of DNA Elements), showed substantial overlap of 5hmC reads with histone modifications marking active transcription.We identified 183 genes containing differential 5hmC in gene bodies at 5% false discovery rate (p<0.001) between MGUS, SMM, and MM patients. Furthermore, 5hmC features in cfDNA showed the potential of separating MM patients at the time of diagnosis according to relapse status (39 genes, p<0.001). Many of these genes were previously implicated in MM, including those found to be differentially methylated (e.g., SYTL1, PSG1, GDF2, DNMT3A, GRP84) in a previous study of CD138+ cells from premalignant MGUS, early, and advanced stages of MM, and healthy control (Heuck CJ, 2013). We are profiling additional ~200 plasma samples from MM patients to 1) validate these preliminary findings, and 2) evaluate independent prognostic value of 5hmC in cfDNA controlling for established prognostic indices. These results will be presented at the ASH meeting.Conclusion: These preliminary findings suggest that 5hmC signatures in cfDNA differ 1) between MM and its two precursor conditions (i.e., MGUS and SMM), and 2) between MM patients by relapse status. [Display omitted] DisclosuresZimmerman:Abbvie: Employment.

Multi-cancer detection and tissue of origin determination based on 5-hydroxymethylcytosine biomarkers in circulating cell-free DNA.

3123 Background: Early detection may reduce cancer mortality. Systematic screening programs are available only for a limited number of cancers (e.g., colorectal cancer). The majority of common cancers are detected after the onset of signs and symptoms, making treatment difficult or less effective. We describe here a multi-cancer epigenetic approach for simultaneous cancer detection of common cancers (̃70% of adult cancers) and determination of tissue of origin (TOO) using circulating cell-free DNA (cfDNA) from plasma. Methods: A total of 2241 cancer cases, including patients with newly diagnosed primary colorectal, gastric, esophageal, liver, lung, and breast cancer (stages I-III or equivalent) and 2289 non-cancer controls were recruited from participating hospitals in China. Study participants were randomly assigned into a training set (70%) and a testing set (30%), and patients were matched for cancer types and stages. Plasma samples were collected before radical treatment or surgery. The 5hmC-Seal, a highly sensitive chemical labeling technique, was used to profile genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA from ̃5mL of plasma per person, followed by the next-generation sequencing, data summarization at gene-level, and normalization. We applied the elastic net regularization to establish a predictive rule based on the multivariable logistic regression model for cancer detection in the training set as well as a multiclass classification model for determining TOO. The final solution for simultaneous cancer detection and TOO determination was established by integrating the 5hmC-based models and protein markers (e.g., AFP). Overall sensitivity and specificity were computed and reported in the testing set of 670 cancer cases and 686 non-cancer controls. Results: For the primary scenario (i.e., stages I-III or equivalent), at specificity of 95%, the overall sensitivity achieved 79.3% for detecting a cancer patient in all six cancer types in the testing set, except stage I lung cancer, for which the multi-cancer detection solution showed a sensitivity of 51%. Notably, for individuals with a negative result from conventional protein markers (e.g., AFP, CEA), the 5hmC-only models showed 67.6% sensitivity at 98.2% specificity in the testing set, representing significant improvement. In the testing set, among the 500 cancer patients who were detected from the multi-cancer detection solution, 435 patients were assigned a TOO; of those, 362 (83.2%) TOO were correctly determined. Conclusions: The 5hmC-Seal in cfDNA shows the potential as a non-invasive tool that could be integrated into a screening program for simultaneous detection of common cancers and TOO localization. This approach can be expanded to additional cancer types and is currently undergoing validation in prospectively recruited cohorts.

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

SummaryWe developed a cost sensitive isotope labelling procedure using a fed‐batch fermentation method and tested its efficiency producing the 15N‐, 13C‐ and 15N/13C‐labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM‐IRRAS and AFM measurements, that the air–water interface triggers structural rearrangement and promotes the amyloid formation of E5, and thus it is a suitable test protein to work out efficient isotope labelling schemes even for such difficult sequences. E. coli cells expressing the recombinant, ubiquitin‐fused miniprotein were grown in minimal media containing either unlabelled nutrients, or 15N‐NH4Cl and/or 13C‐D‐Glc. The consumption rates of NH4Cl and D‐Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH4Cl: D‐Glc). One‐ and two‐step feeding schemes were custom‐optimized to enhance isotope incorporation expressing five different E5 miniprotein variants. With the currently optimized protocols we could achieve a 1.5‐ to 5‐fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols.

Sensitive Label Privacy Preservation with Anatomization for Data Publishing

Data in its original form, however, typically contain sensitive information about individuals. Directly publishing raw data will violate the privacy of people involed. Consequently, it becomes increasingly important to preserve the privacy of published data. An attacker is apt to identify an individual from the published tables, with attacks through the record linkage, attribute linkage, table linkage or probabilistic attack. Although algorithms based on generalization and suppression have been proposed to protect the sensitive attributes and resist these multiple types of attacks, they often suffer from large information loss by replacing specific values with more general ones. Alternatively, anatomization and permutation operations can de-link the relation between attributes without modifying them. In this paper, we propose a scheme Sensitive Label Privacy Preservation with Anatomization (SLPPA) to protect the privacy of published data. SLPPA includes two procedures, table division and group division. During the table division, we adopt entropy and mean-square contingency coefficient to partition attributes into separate tables to inject uncertainty for reconstructing the original table. During the group division, all the individuals in the original table are partitioned into non-overlapping groups so that the published data satisfies the pre-defined privacy requirements of our ( $\alpha,\beta,\gamma,\delta$ α , β , γ , δ ) model. Two comprehensive sets of real-world relationship data are applied to evaluate the performance of our anonymization approach. Simulations and privacy analysis show our scheme possesses better privacy while ensuring higher utility.

Highly Sensitive Determination of Amino Acids by LC-MS under Neutral Conditions

Peptide drug leads possess unusual structural features that allow them to exert their unique biological activities and ideal physicochemical properties. In particular, these peptides often have D-amino acids, and therefore the absolute configurations of the component amino acids have to be elucidated during the structural determination of newly isolated peptide drug leads. Recently, we developed the highly sensitive labeling reagents D/L-FDVDA and D/L-FDLDA for the structural determination of the component amino acids in peptides. In an LC-MS-based structural study of peptides, these reagents enabled us to detect infinitesimal amounts of amino acids derived from mild degradative analysis of the samples. Herein, we firstly report the improved LC-MS protocols for the highly sensitive analyses of amino acids. Second, two new labeling reagents were synthesized and their detection sensitivities evaluated. These studies increase our understanding of the structural basis of these highly sensitive labeling reagents, and should provide opportunities for future on-demand structural modifications of the reagents to enhance their hydrophobicity, stability, and affinity for applications to specialized HPLC columns.

Application of the highly sensitive labeling reagent to the structural confirmation of readily isomerizable peptides.

Thioamycolamide A (1) is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a D-configured thiazoline, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. It has gained attention for its unique structure, and very recently we reported the total synthesis of 1 via a biomimetic route. The NMR spectra of synthetic 1 agreed with those of natural 1. However, structural identity between peptidic natural and synthetic compounds is often difficult to confirm by comparison of NMR spectra because their NMR spectra vary depending on the conditions in the NMR tube, which often result in the structural misassignment of peptidic compounds. Especially, our total synthesis based on the putative biomimetic route potentially gives 1 as a diastereomixture at the final step. The problem is that the diastereomers of peptidic mid-sized molecules often exhibit similar properties (such as NMR spectra and bioactivities), and their separation procedures are often laborious. Herein we report the structural confirmation of synthetic 1 by the LC-MS-based chromatographic comparison with the use of our highly sensitive labeling reagent L-FDVDA; the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method). This work demonstrated the utility of our highly sensitive labeling reagent for the structural determination of not only scarce natural products but also readily isomerizable synthetic compounds.

Measurement of k0 values for europium, lutetium and iridium at FRM\xa0II with a very well thermalized neutron spectrum

The k0 values of 6 non-1/v nuclides (152Eu, 152mEu, 154Eu, 177Lu, 192Ir and 194Ir) were determined using the extended Høgdahl formalism at the research reactor FRM II with very high f values. Standards were irradiated in 4 channels at different local temperatures between 40 °C and 55 °C measured using temperature sensitive irreversible labels. A good agreement with the recommended values was found for 152Eu, 154Eu and 177Lu using the original g(Tn) factors by Gryntakis, however, the k0 values for 152mEu in this work were 7% higher. New k0 values were also determined using the g(Tn) factors by Van Sluijs. Differences up to 6% were found for Eu isotopes compared with the recommended values. The recommended k0 values for 192Ir and 194Ir could be confirmed using g = 1. The theoretical k0 values for 177Lu were calculated using new nuclear data. They are up to 6% less than the recommended values. The present k0 values determined in this work showed a similar trend. The influence of different g(Tn) factors on the determination of the k0 values was investigated.

Impedimetric Biosensor for Monitoring Complementary DNA from Hepatitis B Virus Based on Gold Nanocrystals

A highly sensitive label free electrochemical impedance biosensor based on gold nanocrystals (AuNCs) for hepatitis B virus (HBV) detection in blood serum was reported. A functional platform for measuring the geno-biosensor sensitivity for detecting HBV DNA based on AuNCs electrode was materialized. The adjusted morphologies of 50–100 nm beaded AuNCs on the In2O3 support layer were materialized and characterized by a field emission scanning electron microscope (FESEM) and a high resolution transmission electron microscope (HRTEM). The bio-sensing measurements were conducted by electrochemical impedance spectroscopy (EIS) approach under redox reactions process. The HBV DNA probe (ssDNA) was immobilized on the AuNCs surface by forming a special bond between ssDNA and gold. The DNA target was diagnosed using ssDNA/AuNCs biosensor through EIS measurements in concentration ranges from 0.1 pM to 0.1 μM with a limit of detection (LOD) of 0.1 fM. The selectivity property of the biosensor was investigated and it could distinctively distinguish the complementary DNA target from the non-complementary DNA, 1-, 2- and 3-mismatch targets. Finally, the capability of the electrode for detection of the HBV in blood serum samples was explored and biosensor showed interesting results in the HBV DNA sensing as a potential candidate for practical applications.

The Hybrid Protein ZZ–OL as an Analytical Tool for Biotechnology Research

The gene of the hybrid protein that encodes the double synthetic fragment proZZ of the immunoglobulin-binding domain of protein A of Staphylococcus aureus and apo-obelin joined by a short linker has been cloned. The corresponding hybrid protein has been obtained by expression in Esсheriсhia coli cells. The protein activated with a substrate (coelenterazine) possesses the bioluminescent Са2+-dependent activity of the photoprotein close to that of recombinant wild-type obelin, and the immunoglobulin-binding ability of protein A. It has been shown that the hybrid can be used as a highly sensitive label to detect antibodies and estimate their affinity and interaction with recombinant proteins, as well as in investigations of other kinds.

Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

A versatile system to record cell-cell interactions.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of the cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

Highly Sensitive Labeling Reagents for Scarce Natural Products.

Scarce natural products that possess unique biological activities have been ideal drug leads for decades. However, their identification and structural determinations are problematic owing to sample amount limitation. Inspired by an extremely rare natural product yaku'amide B (10), highly sensitive labeling reagents that would be powerful tools for scarce natural product chemistry were designed and synthesized in this study. By fusion with the key structural motif for the structural revision of 10, the detection sensitivities of amino acid labeling reagents were drastically enhanced in LC-MS analysis. These advanced labeling reagents enabled the detection of infinitesimal amounts of amino acids and peptide hydrolysates. This sensitivity-enhancement design concept was also applicable to reagents for labeling saccharides and reactivity-guided isolation of electrophilic natural products. Details of these reagents, including their practical preparations and extended applications, are also provided.

Silica-Based Gold Nanoshells: Synthesis and Application in Immunochromatographic Assay

We optimize the procedure for synthesizing gold nanoshells for their use as a sensitive label in an immunochromatographic assay (ICA). An immunochromatographic test system is developed to visually determine the concentration of an early cardio marker, a heart-type fatty acid-binding protein (H-FABP). Visual detection yields the detection limit of the procedure with gold nanoshells to be more 50% lower than that of gold nanoparticles; it is 0.5 ng/mL for H-FABP.

Fabrication of a sensitive label free electrochemical immunosensor for detection of prostate specific antigen using functionalized multi-walled carbon nanotubes/polyaniline/AuNPs.

The aim of this research is to introduce a novel label free electrochemical immunosensor based on glassy carbon electrode (GCE) modified with carboxylated carbon nanotubes (COOH-MWCNTs)/polyaniline (PANI)/gold nanoparticles (AuNPs) for the detection of prostate specific antigen (PSA). The AuNPs were utilized as a connector for PSA antibody immobilization through NH2 groups on antibody. Investigations on modified electrode surface were performed by FT-IR spectrum, scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS) to evaluate the synthesized nanocomposite and modified electrode surface. As a sensitive analytical method for the detection of PSA, differential pulse voltammetry (DPV) was employed in different ranges of antigen concentration, 1.66 ag·mL-1 to 1.3ng·mL-1. In addition, the detection limit was obtained 0.5pg·mL-1, from the linear relationship between antigen concentration log and peak current. Also, the proposed immunosensor was carried out for the determination of PSA in human serum samples, indicating recoveries ranging from 92 to 104%. Finally, it should be noted that the reproducibility and specificity, along with the stability of the present immunosensor were examined, and satisfactory findings were obtained, thus proving it as a promising PSA immunosensor.