Complicated Biological Samples Research Articles

Bisphosphorylated fructose-modified magnetic Zr-Organic framework: A dual-hydrophilic sorbent for selective adsorption of immunoglobulin G

Highly selective adsorption of protein species interested from complicated biological samples is an extremely important prerequisite for in-depth proteomic analysis. Herein, a novel dual-hydrophilic material is fabricated by grafting Zr-based metal-organic framework (Zr-MOF) onto the surface of magnetic microspheres, and subsequent modifying with fructose-1,6-bisphosphate (FBP) molecules by taking advantage of multivalent binding between the phosphate backbone of FBP and Zr centers. Owing to the favorable dual-hydrophilicity derived from the abundant amino groups in Zr-MOF and the hydroxyl groups in FBP molecules, the obtained magnetic microspheres (mMOF-FBP) provide high adsorption efficiency, favorable selectivity and excellent capturing capacity (555.6 mg g−1) towards immunoglobulin G (IgG). The captured IgG could be collected from the mMOF-FBP by using 0.5% (m/v) NH3·H2O as elution reagent with a recovery of 88.4%. The dual-hydrophilic microspheres demonstrate their practicability by selective isolation of IgG from human serum.

Carbon dots as rapid assays for detection of mercury(II) ions based on turn-off mode and breast milk.

In this research, nitrogen co-doped carbon dots were synthesized by solid thermal method with citric acid used as the precursor of carbon, and melamine as nitrogen source. Such carbon dots show high quantum yield of 44%. Furthermore, the native fluorescence of CDs can be reduced by mercury(II), while other metals had no significant influence on fluorescence intensity. During the study, the optimal parameters were selected, such as pH or time for incubation with analyte. Under the optimal conditions, quenching effect caused by mercury ions was evaluated. It was observed that with increasing mercury concentration, the fluorescence of the carbon dots decreased proportionally. The response was characterized by linearity within the range from 2 to 14μM. Moreover, the limit of detection was 0.44μM. It was the first time that human milk was used as a real sample to test the applicability of carbon dots. The study results demonstrated good recovery in the 74-111% range (RSD<6%) As a novel carbon material, CDs show promise for broader applications in analyzing complicated biological samples.

Online preconcentration by electrokinetic supercharging for sensitive determination of berberine and jatrorrhizine in biological samples.

Electrokinetic supercharging, a convenient and powerful online preconcentration technique in capillary electrophoresis, was introduced and evaluated for the determination of two alkaloids, berberine and jatrorrhizine, in mice fecal samples for the first time. The method depended on using a bare fused silica capillary (50 cm × 50 μm i.d.) and applying the voltage of 25 kV with UV detection at 205 nm. Parameters that affect the separation and preconcentration efficiency have been optimized. The optimum conditions used were as follows: background electrolyte consisting of 40mM sodium dihydrogenphosphate containing 30% methanol (v/v); hydrodynamic injection of 20mM KCl (50 mbar × 150 s) as the leading electrolyte; electrokinetic injection of the sample (+15 kV, 120 s) followed by the hydrodynamic injection of 30mM dodecyl trimethyl ammonium chloride (50 mbar × 12 s) as the terminating electrolyte. The results showed that the detection sensitivity of berberine and jatrorrhizine was, respectively, improved up 2740- and 2928-fold compared with normal injection, providing limits of detection lower than 3 ng/mL with good repeatability in areas (relative standard deviation < 3%). In summary, the developed method proved its ability in analyzing trace alkaloids in complicated biological samples.

Bifunctional Magnetic Supramolecular-Organic Framework: A Nanoprobe for Simultaneous Enrichment of Glycosylated and Phosphorylated Peptides.

Protein glycosylation and phosphorylation are two important protein post-translational modifications. Mass spectrometry (MS) has been proved to be a powerful technique in comprehensive characterization of protein glycosylation and phosphorylation; however, the complexity of biological matrices and weak ionization efficiency bring a big challenge. Capturing glycopeptides and phosphopeptides from complicated biological samples is indispensable before MS determinations. In this study, a bifunctional gallium ion immobilized magnetic pertriflated pillar[5]arene supramolecular-organic framework (magOTfP5SOF-Ga3+) was designed for the one-step simultaneous enrichment of glycopeptides and phosphopeptides. Thanks to the abundant sulfonic acid groups, the material owns strong hydrophilicity and leads to hydrophilic interaction chromatography for glycopeptides enrichment. Simultaneously, the high loading amount of gallium ion provides immobilized metal ion affinity for phosphopeptides enrichment. The established platform possesses quick magnetic response performance, high sensitivity (detection limits as low as 0.1 fmol and 0.05 fmol for glycopeptides and phosphopeptides, respectively), and good reusability. In addition, the method was applied to the determination of glycopeptides and phosphopeptides in clinical specimens, cell lysates, and mouse liver tissue samples, demonstrating its highly sensitive and specific glycoproteomics and phosphorproteomics analysis in complex biosamples.

Enabling Molecular Gapping and Bridging on a Biosensing Surface via Electrochemical Cross-Linking and Cleavage.

Protein detection in complicated biological samples requires robust design and a rigorous rinsing process. Many recently developed artificial targeting probes, however, often do not possess antibody-like binding strength that enables them to endure harsh biosensing conditions, and the classic 2-to-1 sandwich binding pattern is unavailable for many targets, often necessitating a complicated indirect signal conversion mechanism. Here, an attempt is made to provide innovative "covalent" solutions to such problems by employing peptide reactivity to form a covalent and robust biosensing structure upon target binding. Both the cross-coupling and cleavage of peptide chains are employed to achieve the classic 2-to-1 binding when only one targeting probe is available. Specifically, a targeting probe against the protein and a signaling probe are coimmobilized onto the sensing interface. The ratio between the two probes and their surface density is modulated so that direct cross-linking between the two immobilized probes is suppressed by the average distance between two such probes. Upon protein binding, the protein molecule may bridge that gap by itself. The signaling probe can carry any motif of signal amplification. And here a Cu-ion-complexed motif, which can exhibit peroxidase-like activity upon electrochemical agitation, is employed multipurposely as the catalyst for cross-linking, cleavage, and signal amplification. Three nonhomologous target proteins can be sensitively quantified in serum-spiked samples and clinical sample detection of one of them is also successful; these results suggest the potential of the proposed method in future clinical applications.

Rapid evaluation of immobilized immunoglobulins using automated mass-segmented ToF-SIMS

Surface interactions largely control how biomaterials interact with biology and how many other types of materials function in industrial applications. ToF-SIMS analysis is extremely useful for interrogating the surfaces of complex materials and shows great promise in analyzing biological samples. Previously, the authors demonstrated that segmentation (between 1 and 0.005 m/z mass bins) of the mass spectral axis can be used to differentiate between polymeric materials with both very similar and dissimilar molecular compositions. Here, the same approach is applied for the analysis of proteins on surfaces, focusing on the effect of binding and orientation of an antibody on the resulting ToF-SIMS spectrum. Due to the complex nature of the samples that contain combinations of only 20 amino acids differing in sequence, it is enormously challenging and prohibitively time-consuming to distinguish the minute variances presented in each dataset through manual analysis alone. Herein, the authors describe how to apply the newly developed rapid data analysis workflow to previously published ToF-SIMS data for complex biological materials, immobilized antibodies. This automated method reduced the analysis time by two orders of magnitudes while enhancing data quality and allows the removal of any user bias. The authors used mass segmentation at 0.005 m/z over a 1-300 mass range to generate 60 000 variables. In contrast to the previous manual binning approach, this method captures the entire mass range of the spectrum resulting in an information-rich dataset rather than specifically selected mass spectral peaks. This work constitutes an additional proof of concept that rapid and automated data analyses involving mass-segmented ToF-SIMS spectra can efficiently and robustly analyze a broader range of complex materials, ranging from generic polymers to complicated biological samples. This automated analysis method is also ideally positioned to provide data to train machine learning models of surface-property relationships that can greatly enhance the understanding of how the surface interacts with biology and provides more accurate and robust quantitative predictions of the biological properties of new materials.

Low cost fabrication of microfluidic paper-based analytical devices with water-based polyurethane acrylate and their application for bacterial detection

This study presents a simple, inexpensive and environment-friendly fabrication strategy for microfluidic paper-based analytical devices which can resist the penetration of surfactant solutions and organic solvents, by using water-based polyurethane acrylate via UV light curing. The filter paper’s barrier created using cured PUA could withstand surfactant solutions (10 wt%, CTAB, SDS and Triton X-100) and organic solvents (methanol, isopropanol, DMF, DMSO, etc). This is very useful for analyzing complicated biological samples on the microfluidic paper-based analytical devices. In addition, the expense of water-based polyurethane acrylate is very cheap (about $8/500 g) and PUA developer is water that is environmental-friendly. To further verify its advantage, we successfully demonstrated the proposed microfluidic devices for detection of E. coli targets in tap water and seawater via colorimetric analysis in a fast and convenient manner. Our results revealed that the linear response to E. coli BL21 was in the range of 104∼109 cfu/mL. The proposed method can effectively avoid the damage for the hydrophobic barriers from the solution even some aggressive liquids, and shows great potential in on-site analysis, environmental monitoring, and food safety.

Bioanalytical methodology for determination of glutamate and aspartate for use in pharmacological sciences with application of Integrated Calibration Method

A new calibration approach based on the adaptation of Integrated Calibration Method (ICM) to consecutive two components analysis in separation science is presented. Consecutive ICM method (C-ICM) was conceptually developed and applied to determination of two excitatory amino acids – glutamate and aspartate in cerebrospinal fluids collected by the use of brain microdialysis from freely-moving animals. Both analytes as a neurotransmitters play an important role in formation of the memory trace, and thus the processes of learning and memory. Due to their low concentration and presence of interferences, considered analytical system – animal brain – was a big challenge. High-performance liquid chromatography (HPLC) with electrochemical detection (ECD) was used in all experimental work. The most important feature of proposed method is integration of interpolative and extrapolative ways to calculate analyte concentration in single calibration procedure, which consequently leads to obtain series of six estimations of analytical result. Comparison of individual estimations with each other allows for a more in-depth analysis of systematic errors. It was proved that C-ICM approach enables diagnosis and compensation of systematic errors induced by occurrence of interference effects and improvement of accuracy of analytical results. Most of all, it was demonstrated that application of this method is efficient and useful analytical tool in analysis of complicated biological samples in pharmacology and neuroscience.

A composite consisting of sulfo-functionalized magnetic graphene and mesoporous silica for extraction of metformin and glimepiride prior to their determination by liquid chromatography tandem mass spectrometry.

A new sorbent was synthesized for restricted-access matrix solid phase extraction (RAM-SPE) of the diabetes drugs metformin (MET) and glimepiride (Glim). Mesoporous silica layers were placed on Fe3O4-magnetized graphene modified with sulfo-functionalized pore walls (denoted as magG@mSiO2-SO3H composites). The composites have a large specific surface (173m2·g-1), appropriate pore sizes (typically 3.7nm), and sulfo-functionalized pore walls. Magnetic separation can be accomplished within 10s. The unique properties of the composites allow both MET and Glim to be selectively and quickly extracted from plasma sample. Following magnetic separation and elution by 50% aqueous acetonitrile with 4% ammonium solution, the two drugs were quantified by LC-MS/MS analysis. The assay has high selectivity, good linearity (2.5-4000ng•mL-1 for MET and 0.02-1600ng•mL-1 for Glim), a low detection limit (as low as 60pg•mL-1 for MET and 1pg•mL-1 for Glim), excellent recovery (above 92.2%), and good precision (RSDs <12%). The method was successfully applied in a pharmacokinetic study in beagle dogs. Graphical abstract Schematic representation of the synthesis of sulfo-functionalized magnetic graphene/mesoporous silica composites, giving a material of type magG@mSiO2-SO3H. Results showed its great potential as a feasible and alternative adsorbent for the selective extraction of MET and Glim from complicated biological samples.

Selective separation of bovine hemoglobin using magnetic mesoporous rare-earth silicate microspheres

Selective separation of heme proteins and peptides from complicated biological samples before comprehensive identification and characterization of intact biomolecules which are low stoichiometry is indispensable for ongoing proteomics. Here, three magnetic mesoporous rare-earth silicate (rare-earth = Er, Tm, Yb) microspheres prepared by facile solvothermal method were used as novel adsorbents for the selective isolation of bovine hemoglobin (BHb), and magnetic mesoporous ytterbium silicate showed apparent adsorption efficiency in isolating BHb with a adsorption capacity of 304.4 mg/g after the comparison. The retained BHb could be eluted by using Na2CO3 as stripping reagent. Meanwhile, Circular dichroism spectra illustrated that the microspheres posed no effect on the secondary structure of BHb. The resultant magnetic particles were characterized by transmission electron microscope, scanning electron microscope, x-ray powder diffraction, vibrating sample magnetometer, Fourier transform infrared spectroscopy, N2 adsorption−desorption isotherm and zeta potential. The as-prepared magnetic microspheres showed high specificity for the separation of BHb from bovine plasma as corroborated by SDS-PAGE and MALDI-TOF MS analysis, which would be expected to possess potential application in isolation of other heme proteins in complex biological samples.

A “turn-off” SERS aptasensor based DNAzyme-gold nanorod for ultrasensitive lead ion detection

It is great significance to precisely monitor lead (II) ions (Pb2+) for environment protection and human health monitoring. We designed a sensitive detection strategy for sensitive and selective determination of Pb2+, based on a Pb2+-specific DNAzyme as the catalytic unit, Cy3-labeled DNA modified gold nanorods (AuNRs) as SERS reporter. Firstly, AuNRs surface were employed as a platform for the immobilization of thiolated probe DNA, and then hybridized with DNAzyme catalytic beacons. By taking advantage of DNAzyme digest, a molecular beacon, causes a “turn-off” SERS signal by disrupting the labeled probes. Under the optical conditions, the DNAzyme-AuNRs sensor system exhibited high sensitivity, acceptable stability and reproducibility with a wide linear range from 0.5 to 100 nM (R2 = 0.9973), and an ultra-low detection limit of 0.01 nM. The proposed strategy has additional advantages of being less time-consuming, low-cost and remote query, and avoids the interference of some metals such as Fe3+, Cd2+, Ba2+, Cu2+, Zn2+. The SERS biosensor system has been successfully applied for detecting Pb2+ in real samples with a satisfactory result. The result indicated that the proposed sensing strategy not only enriches SERS platform of monitoring Pb2+ but also exhibits potential for the point-of-care diagnostic application of the clinical screening in complicated biological samples.

Detection of DNA 3'-phosphatase activity based on exonuclease III-assisted cascade recycling amplification reaction

DNA 3'-phosphatase is an essential enzyme, which plays a pivotal role in repairing DNA damage. The peculiar activity of DNA 3'-phosphatase has been proved to associate with a variety of human pathologies. Therefore, sensitive determination of DNA 3'-phosphatase is necessary for clinical diagnosis and therapy. Here, we develop a simple, sensitive, and specific fluorescent biosensor including three DNA chains of hairpin DNA1, hairpin DNA2 and fluorescence probe DNA (FP) for detecting the activity of DNA 3'-phosphatase. First, biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic beads (MB) to get MB-DNA1. DNA 3'-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups, which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment (tDNA1). Next, two cyclic amplification reactions are designed. In cycle I, the tDNA1 hybridizes with the hairpin DNA2, which leads the hairpin structure of DNA2 opened and the fluorescence signal of 6-carboxy-fluorescein (FAM) labeled on hairpin DNA2 turned on. This cyclic reaction is amplified by exonuclease III (Exo III). At the same time, the trigger DNA2 fragment (tDNA2) is obtained. In cycle II, similarly, the tDNA2 hybridizes with FP. Thus, the fluorescence signal of FAM labeled on FP released, which multiplies with the fluorescence signal from cycle I. Finally, this strategy is applied to determine two typical DNA 3'-phosphatases including T4 polynucleotide kinase (T4 PNK) and alkaline phosphatase (ALP) with the detection limit (LOD) of 0.0033 and 0.00037 U/mL, respectively. The method provides a promising platform to evaluate the DNA 3'-phosphatase activity in the complicated biological samples and can be potentially applied in the relevant fields such as biomedical research, drug discovery and clinical diagnosis.

Rational Design of a Long-Wavelength Fluorescent Probe for Highly Selective Sensing of Carboxylesterase 1 in Living Systems.

Rational design of practical probes with excellent specificity and improved optical properties for a particular enzyme is always a big challenge. Herein, a practical and highly specific fluorescent probe for carboxylesterase 1 (CES1) was rationally designed using meso-carboxyl-BODIPY as the basic fluorophore based on the substrate preference and catalytic properties of CES1. Following molecular docking-based virtual screening combined with reaction phenotyping-based experimental screening, we found that MMB (probe 7) exhibited the optimal combination of sensitivity and specificity toward human CES1 in contrast to other ester derivatives. Under physiological conditions, MMB could be readily hydrolyzed by CES1 and release MCB; such biotransformation brought great changes in the electronic properties at the meso position of the fluorophore and triggered a dramatic increase in fluorescence emission around 595 nm. Moreover, MMB was cell membrane permeable and was successfully applied to monitor the real activities of CES1 in various biological samples including living cells, tissue slices, organs, and zebrafish. In summary, this study showed a good example for constructing specific fluorescent probe(s) for a target enzyme and also provided a practical and sensitive tool for real-time sensing of CES1 activities in complicated biological samples. All these findings would strongly facilitate high-throughput screening of CES1 modulators and the studies on CES1-associated physiological and pathological processes.

Postsynthetic Functionalization of Zr4+-Immobilized Core-Shell Structured Magnetic Covalent Organic Frameworks for Selective Enrichment of Phosphopeptides.

Chemical modification of covalent organic frameworks (COFs) is indispensable for integrating functionalities of greater complexity and accessing advanced COF materials suitable for more potential applications. Reported here is a novel strategy for fabricating controllable core-shell structured Zr4+-immobilized magnetic COFs (MCNC@COF@Zr4+) composed of a high-magnetic-response magnetic colloid nanocrystal cluster (MCNC) core, Zr4+ ion-functionalized two-dimensional COFs as the shell by sequential postsynthetic functionalization and, for the first time, the application of the MCNC@COF@Zr4+ composites for efficient and selective enrichment of phosphopeptides. The as-prepared MCNC@COF@Zr4+ composites possess regular porosity with large surface areas, high Zr4+ loading amount, strong magnetic responsiveness, and good thermal/chemical stability, which can serve as an ideal adsorbent for selective enrichment of phosphopeptides and simultaneous size exclusion of biomacromolecules, such as proteins. The high detection sensitivity (10 fmol) together with the excellent recovery of phosphopeptides is also obtained. These outstanding features suggest that the MCNC@COF@Zr4+ composites are of great benefit for pretreatment prior to mass spectrometry analysis of phosphopeptides. In addition, the performance of the developed approach in selective enrichment of phosphopeptides from the tryptic digests of defatted milk and directly specific capture of endogenous phosphopeptides from human serum gives powerful proof for its high selectivity and effectiveness in identifying the low-abundance phosphopeptides from complicated biological samples. This study not only provides a strategy for versatile functionalization of magnetic COFs but also opens a new avenue in their use in phosphoproteome analysis.

Highly Sensitive Electrochemical Detection of Hydrogen Peroxide Based on Polyethyleneimine-Au Nanoparticles-Zinc Protoporphyrin

A novel electrochemical biosensor for sensitive detection of hydrogen peroxide (H2O2) was developed on the basis of polyethyleneimine-Au nanoparticles-zinc protoporphyrin (PEI-AuNPs-ZnPP). In this approach, PEI-AuNPs-ZnPP was prepared by coupling carboxyl of ZnPP with amino of PEI-AuNPs. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were used to characterize the formation of PEI-AuNPs-ZnPP. Under optimal conditions, this strategy showed a good sensitivity for H2O2 detection with a low detection limit of 0.0861 pM. What's more, this biosensor can be successfully applied for the measurement of H2O2 in human serum samples. These results suggested that PEI-AuNPs-ZnPP could be a good candidate for H2O2 detection in complicated biological samples.

Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies.

The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.

Nickel Nanowires Combined with Surface-Enhanced Raman Spectroscopy: Application in Label-Free Detection of Cytochrome c-Mediated Apoptosis.

Intrinsic properties of nickel have enabled its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity toward Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid label-free Cyt c quantification can be achieved by surface-enhanced Raman spectroscopy with a limit of detection of 1 nM and long concentration linear range (1 nM-1 μM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.

Polydopamine nanoparticle-based multicolor proximity immunoassays for ultrasensitive, multiplexed analysis of proteins using isothermal quadratic amplification

Simple, rapid and selective methods for simultaneous determination of ultralow amount of multiple protein biomarkers in complicated biological samples are in great demand for early-stage diagnostics and biomedical research. Herein, we present a novel polydopamine nanoparticle (PDANP)-based multicolor fluorescent proximity immunoassay for ultrasensitive and multiplexed analysis of proteins in a single solu- tion using isothermal quadratic amplification (IQA). The assay system consists of DNA tethered-antibody pairs for different protein targets, different fluorophore-labeled DNA hairpin probes, DNA polymerase, T7 exonuclease and PDANPs. The DNA tethered- antibodies and fluorophore-labeled hairpin probes can coexist stably, and fluorophore- labeled DNA hairpin probes are adsorbed onto the PDANPs, leading to the significant quenching of the fluorescence of all fluorophores. When target proteins enter into the system, each pair of DNA tethered-antibodies simultaneously binds to a specific pro- tein target and forms the proximate complexes via proximity-dependent DNA hybridi- zation. The formed proximate complexes can then unfold the hairpin structures of the respective probes, leading to DNA polymerase/T7 exonuclease-catalyzed recycling of both proximate complexes and DNA strands to achieve IQA. This IQA results in the di- gestion of many fluorophore-labeled hairpin probes, causing a substantial increase in the fluorescence of corresponding fluorophores for detection of target proteins. As a proof of concept, we demonstrate that a three-color proximity immunoassay can si- multaneously detect prostate-specific antigen (PSA), carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) with ultralow attomolar detection limits, high specificity and 6-log dynamic range. Furthermore, the application of this assay for direct and simulta- neous determination of multiple proteins in clinical sera has also been demonstrated. Our newly proposed assay can be expected to provide a generic method platform for multiplexed analysis of a variety of low-abundance proteins.

Branched DNA Junction-Enhanced Isothermal Circular Strand Displacement Polymerization for Intracellular Imaging of MicroRNAs.

The microRNA profiles within living cells are informative for diagnosis and prognosis of human cancers. In the present work, we developed a new sensing strategy based on branched DNA junction-enhanced isothermal circular strand displacement polymerization (B-ICSDP) for the detection and intracellular imaging of microRNAs in living cells of interest. A circular DNA template consisting of three repetitive fragments serves as the scaffold for the self-assembly of sophisticated signaling probes, resulting a shrunk branched DNA junction. Target microRNA triggers the opening of molecular beacon, not only restoring the quenched fluorescence but also activating a circular polymerization-based strand displacement reaction. Thus, patulous branched DNA junction is abundantly formed, generating the amplified signal. It is noteworthy that great heaps of branched product assemblies can be also achieved in living cells, and the intracellular enzymatic assembly based strategy is able to be used to recognize specific microRNA-expressed cancer cells. Moreover, different microRNAs coexisting in the same living cells can be simultaneously screened without any interference from each other by confocal laser scanning microscopy. The measured data from confocal fluorescence imaging of different cancer cells demonstrates that the B-ICSDP-based system is a promising alternative for in vivo analysis of microRNAs in complicated biological samples.

Enrichment of Phosphorylated Peptides with Metal-Organic Framework Nanosheets for Serum Profiling of Diabetes and Phosphoproteomics Analysis.

Capturing phosphopeptides from complicated biological samples is essential for the discovery of new post-translational modification sites and disease diagnostics. Although several two-dimensional (2-D) materials have been used for phosphopeptides capturing, metal-organic framework (MOF) nanosheets have not been reported. The Ti-based MOF nanosheets have well-defined 2-D morphology, high density of active sites, large surface area, and an ultrathin structure. Phosphopeptides can be efficiently extracted and superior detection limits of 0.1 fmol μL-1 can be achieved even for an extremely low molar ratio of phosphoprotein/nonphosphoprotein (1:10000) mixtures. The selectivity over nonphosphopeptides can be enhanced further by pretreatment with a 10 mM salt solution (β-glycerophosphate disodium, NaCl, or KCl). The performance of 2-D Ti-based MOF nanosheets is much better than Zr-based MOF (Zr-BTB) nanosheets or any other Ti-based 3-D MOF counterpart, such as MIL-125 and NH2-MIL-125. The nanosheets were used for in situ isotope labeling for abnormally regulated phosphopeptides analysis from serum samples of type 2 diabetes patients. The relative quantitative results showed that three of the phosphorylated fibrinogen peptides A (FPA, DpSGEGDFLAEGGGV, DpSGEGDFLAEGGGVR, and ADpSGEGDFLAEGGGVR) were down-regulated, while the other isoform (ADpSGEGDFLAEGGGV) was up-regulated in the serum samples of type 2 diabetes patients compared with those of healthy volunteers. Finally, proteomics analysis showed selective enrichment of phosphopeptides with 2-D Ti-based MOF nanosheets from real samples, including tryptic digests of mouse brain neocortex lysate, mouse spinal cord lysate, and mouse testis lysate, followed by LC-MS/MS analysis. Total numbers of 2601, 3208, and 2866 phosphopeptides were successfully identified from the three samples, respectively. The 2-D Ti-based MOF nanosheets significantly improved sample preparation for mass spectrometric analysis in phosphopeptides and phosphoproteomics research.