Real Complex Biological Samples Research Articles

Sensing multiplex microRNAs using dual-functional fork-type-track DNA nanomachine on optical fiber

We propose a simple and robust biosensing platform which can be used for accurate determination of multiplex microRNA targets in biological samples. We develop the sensor by immobilizing a novel DNA nanomachine system on the surface of a gold-nanoparticles-modified optical fiber, which contains a uniquely designed dual-functional fork-type track chain with a FAM-labeled DNAzyme recognition site on one branch and a rolling-loop-amplification (RCA) primer on the other. The strategy allows two amplification strategies, i.e., DNA walking and RCA, to be easily achieved as the target microRNAs are in the sample to initiate the corresponding reactions. Two different microRNAs (miRNA-21 and miRNA-375 in the study) are independently and accurately determined without interference from each other, by respectively measuring fluorescence and chemiluminescence signals generated from DNA walking and RCA. The results show that the method is capable for multiplex micorRNA assays with high sensitivity (limit-of-detection 0.21 pM for miRNA-21 and 0.018 pM for miRNA-375) and high selectivity, and the sensor exhibits good repeatability and storage stability. The method successfully determines microRNAs in MCF-7, Hela and MCF-10A cells, showing its ability to analyze real complex biological samples.

Low fouling electrochemical sensing interfaces based on Bovine serum albumin hydrogel for the detection of protein biomarkers in human serum

The development of electrochemical biosensor capable of detecting specific biomarkers in real complex biological samples remains a challenge significantly relevant to a range of disease diagnostic applications. Herein an antifouling electrochemical biosensor suitable for biomarker detection in human serum was constructed based on protein hydrogels. A typical protein bovine serum albumin (BSA) was crosslinked with glutaraldehyde to form a hydrogel, which was freeze-dried and re-swollen to exhibit excellent antifouling capability. The rheological testing revealed that the prepared BSA hydrogel possessed good elasticity with a critical strain value of about 600 %, which is important for the construction of stable hydrogel-based biosensors. Following the immobilization of CA125 antibodies onto the BSA hydrogel modified electrode, an electrochemical biosensor for the detection of CA125 was constructed, with a linear range of 0.1 ∼ 1000 U mL−1 and a limit of detection of 0.026 U mL−1. Owing to the unique properties of the BSA hydrogel, the biosensor exhibited good stability and excellent antifouling performances, and it was able to detect CA125 in human serum with acceptable accuracy. The strategy of constructing low fouling biosensors based on hydrogels prepared from BSA with biocompatibility and widespread availability represented a very promising advance along the road to develop biosensing devices with robust operation in complex biological fluids.

A Facile Glycerol-Assisted Synthesis of Low-Cu2+-Doped CoFe2O4 for Electrochemical Sensing of Acetaminophen.

This work devised a simple glycerol-assisted synthesis of a low-Cu2+-doped CoFe2O4 and the electrochemical detection of acetaminophen (AC). During the synthesis, several polyalcohols were tested, indicating the efficiency of glycerin as a cosolvent, aiding in the creation of electrode-modifier nanomaterials. A duration of standing time (eight hours) before calcination produces a decrease in the secondary phase of hematite. The synthesized material was used as an electrode material in the detection of AC. In acidic conditions (pH 2.5), the limit of detection (LOD) was 99.4 nM, while the limit of quantification (LOQ) was found to be (331 nM). The relative standard deviation (RSD), 3.31%, was computed. The enhanced electrocatalytic activity of a low-Cu2+-doped CoFe2O4-modified electrode Cu0.13Co0.87Fe2O4/GCE corresponds extremely well with its resistance Rct, which was determined using the electrochemical impedance spectroscopy (EIS) technique and defined its electron transfer capacity. The possibility of a low-Cu2+-doped CoFe2O4 for the electrochemical sensing of AC in human urine samples was studied. The recovery rates ranging from 96.5 to 101.0% were obtained. These findings suggested that the Cu0.13Co0.87Fe2O4/GCE sensor has outstanding practicability and could be utilized to detect AC content in real complex biological samples.

Ratiometric Antifouling Electrochemical Biosensors Based on Multifunctional Peptides and MXene Loaded with Au Nanoparticles and Methylene Blue.

A universal strategy for the construction of ratiometric antifouling electrochemical biosensors was developed based on multifunctional peptides and 2D nanomaterial MXene loaded with gold nanoparticles (AuNPs) and methylene blue (MB). The nanocomposite of MXene loaded with AuNPs and MB (MXene-Au-MB) exhibited excellent conductivity, where the AuNPs were able to capture biomolecules containing sulfhydryl terminus, and the MB molecules were used to generate electrochemical signal. The MXene-Au-MB was fixed on the electrode surface by Nafion, and the anchored peptide captured the electrochemical signal probe carboxyl-modified ferrocene (Fc) to construct an electrochemical biosensor. The multifunctional peptide containing the anchoring, antifouling, and recognizing sequences endowed the sensing surface not only the assaying function but also the capability to resist nonspecific adsorption from complex samples. In the biosensing system, with the increase in the target concentration, the electrochemical signal of MB remained constant, whereas the electrochemical signal of Fc gradually decreased, and the ratiometric detection strategy greatly improved the accuracy of the biosensor. In the presence of a model target prostate-specific antigen (PSA), the recognizing sequence was recognized and cleaved, and the ratiometric signal of Fc and MB indicated the concentration of PSA accurately and sensitively, with a detection range from 5 pg/mL to 10 ng/mL and a limit of detection of 0.83 pg/mL. Electrochemical biosensors based on the MXene-Au-MB and multifunctional peptides possessed high selectivity, accuracy, and sensitivity even in real complex biological samples because of the excellent antifouling ability of the peptide. More importantly, the assaying of other targets can be easily realized with a similar biosensing strategy by changing the recognition sequence of the multifunctional peptide, and the detection of thrombin (TB) has also been achieved in this work.

Selective Enrichment of Low-abundance Compounds in a Mixture by Capillary Electrophoresis

Recently, we developed a new approach for the selective enrichment of low-abundance compounds in biological samples by capillary electrophoresis. As a model test, the low-abundance compound lysozyme was successfully fractionated from a mixture containing high-abundance compound BSA (1:4500) using a custom-made apparatus. The feasibility of this approach for real complex biological samples was verified by rat serum, wherein three low-abundance proteins with high charge/mass ratios were detected.

Efficient ratiometric fluorescence probe utilizing silicon particles/gold nanoclusters nanohybrid for "on-off-on" bifunctional detection and cellular imaging of mercury (II) ions and cysteine.

An effective ratiometric fluorescent probe based on silicon particles/gold nanoclusters (SiNPs/AuNCs) nanohybrid has been fabricated and applied to be a "on-off-on" switch sensing platform for detection of Hg2+ and cysteine. In this elaborated sensing platform, the SiNPs just acted as internal reference signal, providing a build-in correction for background interferences and environmental effects, to which the AuNCs as a signal report unit for Hg2+ response was covalently grafted by amidation reaction. The fluorescence intensity of SiNPs/AuNCs could be effectively quenched upon adding Hg2+, accompanied with an easily distinguishable fluorescent color change. The ratiometric fluorescence signal (F649/F511) of the established nanoprobe was linearly proportional to the concentration of Hg2+ ranging from 0.02 to 24μM with a low detection limit of 5.6nM, which is below the guideline value of Hg2+ in drinking water set by the World Health Organization. Interestingly, upon addition of cysteine, the Hg2+-quenched fluorescence intensity was recovered gradually. Furthermore, the approach developed has also been utilized for Hg2+ detection in real complex biological samples with satisfactory results. More importantly, benefiting from the good water-solubility and excellent biocompatibility, this nanoprobe can monitor the intracellular Hg2+ and cysteine in living cells, indicating its potential applications in advanced biosensing and bioimaging.

Development of amphiphile 4-armed PEO-based Ti4+ complex for highly selective enrichment of phosphopeptides

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4μ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4μ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol β-casein, the 4μ-PEO-Ti4+ performed the best property with starting material of 99–132 μg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4μ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4μ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of β-casein from a mixture with BSA of 1000-fold abundant. The 4μ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4μ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4μ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.

CliqueMS: a computational tool for annotating in-source metabolite ions from LC-MS untargeted metabolomics data based on a coelution similarity network.

MotivationThe analysis of biological samples in untargeted metabolomic studies using LC-MS yields tens of thousands of ion signals. Annotating these features is of the utmost importance for answering questions as fundamental as, e.g. how many metabolites are there in a given sample.ResultsHere, we introduce CliqueMS, a new algorithm for annotating in-source LC-MS1 data. CliqueMS is based on the similarity between coelution profiles and therefore, as opposed to most methods, allows for the annotation of a single spectrum. Furthermore, CliqueMS improves upon the state of the art in several dimensions: (i) it uses a more discriminatory feature similarity metric; (ii) it treats the similarities between features in a transparent way by means of a simple generative model; (iii) it uses a well-grounded maximum likelihood inference approach to group features; (iv) it uses empirical adduct frequencies to identify the parental mass and (v) it deals more flexibly with the identification of the parental mass by proposing and ranking alternative annotations. We validate our approach with simple mixtures of standards and with real complex biological samples. CliqueMS reduces the thousands of features typically obtained in complex samples to hundreds of metabolites, and it is able to correctly annotate more metabolites and adducts from a single spectrum than available tools.Availability and implementation https://CRAN.R-project.org/package=cliqueMS and https://github.com/osenan/cliqueMS.Supplementary information Supplementary data are available at Bioinformatics online.

A magnetic hydrazine-functionalized dendrimer embedded with TiO2 as a novel affinity probe for the selective enrichment of low-abundance phosphopeptides from biological samples

Dendrimers exhibit tunable terminal functionality and bio-friendly nature, making them of being promising materials for applications in the field of separation and enrichment. In this work, we prepared magnetic hydrazide-functionalized poly-amidoamine (PAMAM) dendrimer embedded with TiO2 for the enrichment of phosphopeptides. The novel affinity probe possessed superparamagnetism, realizing its rapid separation from sample solution. Electrostatic attraction and hydrogen bonding existed between PAMAM and phosphopeptides while Lewis acid-base interaction was originated between TiO2 and the targets. The combined synergistic strength of multiple binding interactions contributed to the highly selective enrichment of phosphopeptides. The specificity for the capture of phosphopeptides was reflected in quantities as low as 1:1000 mass ratio of phosphopeptides to non-phosphopeptides. The detection limit of β-casein digests was low to 0.4 fmol, indicating the high sensitivity of the developed method. Fifteen and four phosphopeptides could be selectively captured from non-fat milk digests and human serum samples, which further confirmed the great potential of the affinity probe in the extraction of low-abundance phosphopeptides from real complex biological samples.

Dual-Metal Centered Zirconium-Organic Framework: A Metal-Affinity Probe for Highly Specific Interaction with Phosphopeptides.

The highly specific affinity between probes and phosphopeptides is the fundamental interaction for selective identification of phosphoproteomes that uncover the mechanisms of signal transduction, cell cycle, enzymatic regulation, and gene expression in biological systems. In this study, a metal-affinity probe possessing both interactions of metal oxide affinity chromatography (MOAC) and immobilized metal ion affinity chromatography (IMAC) was facilely prepared by immobilizing zirconium(IV) on a zirconium-organic framework of UiO-66-NH2, which holds dual-metal centers of not only the inherent Zr-O cluster but also the immobilized Zr(IV) center. This dual-metal centered zirconium-organic framework (DZMOF) demonstrates as a highly specific metal-affinity probe toward the extraction of phosphopeptides due to the metal-affinity interactions of MOAC and IMAC toward either mono-phosphorylated or multi-phosphorylated peptides. The binding energies of zirconium 3d5/2 and 3d3/2 in this DZMOF are 183.07 and 185.47 eV, respectively, which are higher than those of the intact UiO-66-NH2 (182.84 and 185.17 eV, respectively), confirming the higher metal-affinity interaction between the DZMOF and phosphopeptides. This high metal-affinity probe presents an unprecedented strong performance in anti-nonspecific interference during the capturing of phosphopeptides of β-casein with the molar ratio of β-casein vs bovine serum albumin up to ca. 1:5000. The enrichment of phosphopeptides from a human saliva sample by DZMOF further confirms the great potential of DZMOF in the extraction of low-abundance phosphopeptides for real complex biological samples.

A simple peak detection and label-free quantitation algorithm for chromatography-mass spectrometry.

BackgroundLabel-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.ResultsWe developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.ConclusionsAB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users.

Magnetic MSP@ZrO₂ microspheres with yolk-shell structure: designed synthesis and application in highly selective enrichment of phosphopeptides.

Magnetic yolk-shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via a two-step controlled "sol-gel" approach for the first time. First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid matrix via a strong hydrogen-bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form a yolk-shell structure, which has better dispersibility and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk-shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD, and FT-IR characterization. By taking advantage of the unique properties, the yolk-shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk-shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk-shell MSP@ZrO2-TiO2.

Preparation of cysteine-click maltose modified silica as a hydrophilic interaction liquid chromatography material for the enrichment of glycopeptides

Because of the low abundance of glycoprotein and glycopeptide in complex biological samples, it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research. Herein, a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO2). The developed hydrophilic silica was packed into a solid phase extraction (SPE) column, and applied to the highly selective enrichment and identification of N-linked glycopeptides. The Cys-Mal@SiO2 demonstrated better identification capability over Cys@SiO2, Mal@SiO2 and commercial hydrophilic interaction liquid chromatography (HILIC) in glycopeptide enrichment due to the synergistic effect of the two kinds of hydrophilic molecules. In the selective enrichment of tryptic digest from human immunoglobulin G, glycopeptides with higher signal-to-noises were detected by Cys-Mal@SiO2. In addition, 1551 unique glycopeptides with 906 N-glycosylation sites from 466 different N-linked glycoproteins were identified from the proteins extracted from mouse liver after the enrichment with Cys-Mal@SiO2. In contrast, the numbers of identified glycopeptides, glycoproteins and N-glycosylation sites identified by Cys@SiO2 were 211, 67, 127 respectively less than by Cys-Mal@SiO2, and the corresponding numbers were 289, 76, 193 by Mal@SiO2. These results showed that the developed Cys-Mal@SiO2 is a promising affinity material for N-glycoproteomics research of real complex biological samples.

Automated screening for metabolites in complex mixtures using 2D COSY NMR spectroscopy

One of the greatest challenges in metabolomics is the rapid and unambiguous identification and quantification of metabolites in a biological sample. Although one-dimensional (1D) proton nuclear magnetic resonance (NMR) spectra can be acquired rapidly, they are complicated by severe peak overlap that can significantly hinder the automated identification and quantification of metabolites. Furthermore, it is currently not reasonable to assume that NMR spectra of pure metabolites are available a priori for every metabolite in a biological sample. In this paper we develop and report on tests of methods that assist in the automatic identification of metabolites using proton two-dimensional (2D) correlation spectroscopy (COSY) NMR. Given a database of 2D COSY spectra for the metabolites of interest, our methods provide a list sorted by a heuristic likelihood of the metabolites present in a sample that has been analyzed using 2D COSY NMR. Our models attempt to correct the displacement of the peaks that can occur from one sample to the next, due to pH, temperature and matrix effects, using a statistical and chemical model. The correction of one peak can result in an implied correction of others due to spin–spin coupling. Furthermore, these displacements are not independent: they depend on the relative position of functional groups in the molecule. We report experimental results using defined mixtures of amino acids as well as real complex biological samples that demonstrate that our methods can be very effective at automatically and rapidly identifying metabolites.