Dual-modal analysis of cis-diols in traditional Chinese medicine via boronic acid incorporated metal organic frameworks.

In this study, a boronic acid incorporated metal organic frameworks (inBA-MIL-100) were prepared via metal-ligand-fragment co-assembly strategy. The prepared frameworks can be served either as enrichment sorbent or SALDI-MS matrix for cis-diol containing molecules. Thus, a dual-modal analysis of cis-diols in traditional Chinese medicine has been established. Several significant advantages of the proposed strategy have been experimentally demonstrated, including high selectivity, high binding capacity (70 mg/g), good generality (5-250 µg/mL for HPLC based sample preparation, 10-500 ng/mL for SALDI-MS), high sensitivity (LOD: 180 ng/mL for HPLC based sample preparation, 5 ng/mL for SALDI-MS) and reliable quantification (RSD<3 % for HPLC based sample preparation, RSD<12 % for SALDI-MS) performance. Finally, the successful analysis of various cis-diols (active component and mycotoxin) in various Chinese traditional medicine was also achieved.

Structure-Assisted Boronic Acid Implanted Mesoporous Metal-Organic Frameworks for Specific Extraction of cis-Diol Molecules.

cis-Diol-containing molecules, an essential type of compounds in living organisms, have attracted intensive research interest from various fields. The analysis of cis-diol-containing molecules is still suffering from some drawbacks, including low abundance and abundant interference. Metal-organic frameworks (MOFs) have proven to be an ideal sorbent for sample preparation. However, most of the reported MOFs are mainly restricted to a microporous regime (pore size <2 nm), which greatly limits the application. Herein, a facile strategy is established to construction of boronate affinity MOFs via the postsynthetic ligand-exchange process. Owing to the fact that the ligand-exchange process was assisted by the structural integrity of the primitive metal-organic framework and the great compatibility of click chemistry, the obtained EPBA-PCN-333(Fe) is able to realize the maximum maintaining the porosity and crystallinity of the parent material. Several intriguing features of EPBA-PCN-333(Fe) (e.g., excellent selectivity, efficient diffusion, good accessibility, and size exclusion effect) are experimentally demonstrated via a series of cis-diol-containing molecules with different molecular sizes (small molecules, glycopeptides, and glycoproteins). The binding performance of EPBA-PCN-333(Fe) is evaluated by employing catechol as the test molecule (binding capacity: 0.25 mmol/g, LOD: 200 ng/mL). Finally, the real-world applications of EPBA-PCN-333(Fe) were demonstrated by the detection of nucleosides of human urine samples.

Magnetic dendritic mesoporous silica nanoparticles based integrated platform for rapid and efficient analysis of saccharides.

BACKGROUND: As an essential compound in living organism, saccharides have attracted enormous attentions from scientists in various fields. Understanding the distribution of saccharides in various samples is of great scientific importance. However, the low signal response and lack of specific recognition technology of saccharides and the complex matrix of samples make the analysis of saccharides a very challenge task. Thus, the development of a simple and straightforward strategy for the analysis of saccharides would represent a great contribution to the field. RESULTS: In this study, by employing the sulfonyl functionalized magnetic dendritic mesoporous silica nanoparticles as the substrate, we develop an integrated platform for analysis of saccharides. The construction of the platform mainly relied on multi-functional boronic acid, which serves as separation and derivation ligands at the same time. In the general procedure, the boronic acid is first immobilized onto the surface of substrate, then the selective enrichment of saccharides can be realized via boronate affinity separation. Finally, by the rational choice of the solution, we are able to elute the labelled complex (boronic acid-saccharide) from the substrate, which can be direct subjected to HPLC-UV analysis. The reliable precision (<15 %), accuracy (80-100 %), reproducibility (<10 %), improved sensitivity (20x) and limited time-consuming (down to minutes) of the proposed platform are experimentally demonstrated. SIGNIFICANCE AND NOVELTY: The successful quantification of different saccharides (alditols, glucose) in real samples is achieved. The proposed strategy is not only straightforward and fast, but also avoid the requirement of special equipment. With these attractive features, we believe that this strategy will greatly prompt the analysis of saccharides in various samples (eg. food, pharmaceutics and biosamples).

In situ digestion-assisted multi-template imprinted nanoparticles for efficient analysis of protein phosphorylation.

A new general approach called in situ digestion-assisted multi-template imprinting is proposed for preparation of phospho-specific molecularly imprinted nanoparticles. Through the novel templating strategy and controllable imprinting process, imprinted nanoparticles specific to the intact phosphoprotein and its phosphopeptides were synthesized. The prepared imprinted nanoparticles exhibited excellent specificity (cross reactivity < 10%), high affinity (10-6 M), high efficiency (47.5%), and good generality (both intact phosphoprotein and phosphopeptides). We also realized the fine tuning of the recognition at peptide level of the imprinted nanoparticles by adjusting the imprinting time. Based on the selective enrichment of the imprinted nanoparticles, the MS identification of both the intact phosphoprotein (Tau) and phosphopeptides (angiotensin II and peptides of Tau) in real complex samples could be achieved. Therefore, we believe that the in situ digestion-assisted multi-template imprinting strategy holds promising future in both phosphorylation analysis and proteomics applications.

Diboronic acid assisted labeling and separation for highly efficient analysis of saccharides.

In this study, a diboronic acid assisted labeling and separation strategy was proposed for the efficient analysis of saccharides in real samples. The diboronic acid was first synthesized and characterized and then used as chemical labels for saccharides through boronate affinity. After labeling, the product was direct submitted to chromatographic analysis. The whole labeling procedure was organic solvent-free and could be accomplished within 1 h. Furthermore, the separation could be realized on the most commonly used chromatographic techniques (RPLC-UV). Taking glucose as an example, the established strategy provided broad linear dynamics ranges (25-5000 ng/mL) with acceptable correlation coefficients (R2>0.98), substantial sensitivity (LOD: 5 ng/mL), high reproducibility (RSD<6.7%) and excellent accuracy (Recovery: 98.2-99.8%). Finally, different saccharides were successfully analyzed in various complex samples, from tryptic glycans of glycoproteins to monosaccharides in traditional Chinese medicine.

Selective analysis of interferon-alpha in human serum with boronate affinity oriented imprinting based plastic antibody.

Interferons are important biomolecules in human immune system. Cytokine interferon alpha (IFN-α), a type I interferon, is one of the major components of the innate immune response involved in autoimmune diseases. Thus, the analysis of interferons is of great importance for both biological and pharmaceutical purposes. In this work, an IFN-α specific plastic antibody is prepared via boronate affinity oriented surface imprinting. By combing with the magnetic nanoparticles, the imprinted material exhibits several advantages, including strong affinity (Kd: 75.2 nM), high specificity (cross reactivity<25%), excellent efficiency (imprinting efficiency: 44.1%), tolerance to interferences, and easy manipulation. By employing the prepared imprinted material as sorbent for selective enrichment of IFN-α, a good linearity is achieved in the range of 50 ng/mL-10 µg/mL, and the detection and quantifcation limits are 10 ng/mL and 50 ng/mL respectively. The recoveries of this approach are found within 75.8%-82.2% with relative standard deviations of 6.4-9.7%. Furthermore, the IFN-α in spiked human serum is analyzed with acceptable reliability (recovery: 77.3%, RSD: 7.9%). Because of these highly desirable properties, the IFN-α specific plastic antibody can find more applications in medical and pharmaceutical industry.

PEI-assisted boronate affinity magnetic nanoparticle-based SELEX for efficient in vitro evolution of saponin-binding aptamers.

Saponin is a large family of important natural products with various pharmacological activities. Selective enrichment of saponin from complex biological samples is a key step for analysis of saponin. Despite that aptamers have been widely used for selective enrichment, aptamers that can specifically recognize saponins have never been reported. In this study, a facile and efficient SELEX approach was developed for in vitro evolution of saponin-binding aptamers, using PEI-assisted boronate affinity magnetic nanoparticles (p-BA-MNPs) that exhibit highly favorable binding properties as a general affinity platform. As a proof of the principle, ginsenoside Re and Rb1 were employed as two target saponins. Two aptamers towards each target saponin, with dissociation constant at the 10-5 M level, were selected within 6 rounds. An affinity magnetic nanoparticle was constructed by using the selected aptamer as a affinity ligand. The resulting material allowed for the quantitative analysis of ginsenoside Re in real samples with high reliability. The p-BA-MNPs based SELEX is straightforward and generally applicable for a wide range of target saponins, providing a promising aptamer evolution approach for aptamer-based research and pharmaceutical analysis.

Crystalline MOF nanofilm-based SALDI-MS array for determination of small molecules.

A highly oriented crystalline metal-organic framework (MOF) nanofilm array was prepared and used for surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) for determination of small molecules. The MOF nanofilm was characterized using scanning/transmission electron microscopy, X-ray diffraction, and ultraviolet-visible spectroscopy. Different small molecules (anthracene, n-eicosanoic acid, Rhodamine B) were successfully determined by this MOF nanofilm array with limits of detection (LOD) between 0.1-5 ng·mL-1 and limits of quantification (LOQ) between 1 and 10 ng·mL-1. Compared to previously reported MOF-based SALDI-MS, this array exhibits better reproducibility (3.3-5.2%) and recovery (89-105%). The intensity of the MS peak remains the same after 25 repeated cycles. This indicates good repeatability. This MOF nanofilm-based SALDI-MS array can be used for determination of fatty acid and Rhodamine B in real samples with good recovery (83-106%). Graphical abstract Schematic representation of the principle of crystalline MOFs nanofilm-based SALDI-MS array.

Dual boronate affinity nanoparticles-based plasmonic immunosandwich assay for specific and sensitive detection of ginsenosides.

Ginsenoside is a large family of triterpenoid saponins from Panax ginseng with various important biological functions. It is crucial to develop effective analytical approach for qualitative and quantitative analysis of ginsenosides. Herein, a dual boronate affinity nanoparticles-based plasmonic immunosandwich assay has been developed for analysis of ginsenosides. An imprinted Au NPs-coated glass slide was prepared via controllable oriented surface imprinting and used as specific extraction substrate for target molecules. In the meantime, Ag-cored Raman nanotags were used for specific labeling of target molecules. The MIP-based recognitions ensured the specificity of the assay, while enhanced Raman signal derived from the imprinted substrate-target-nanotags sandwich-like complexes provided high sensitivity. The proposed immunosandwich assay exhibited wide linear range (10 ng/mL to 10 µg/mL), high sensitive (LOD: 1.7 ng/mL, LOQ: 5 ng/mL) and good reproducibility (RSD: 8.6%). For real-world applications, successful quantitative analysis of ginsenoside Re in ginseng was performed. Therefore, this dual boronate affinity nanoparticles-based plasmonic immunosandwich assay holds great promise in many important applications such as pharmaceutical analysis.

Preparing molecularly imprinted nanoparticles of saponins via cooperative imprinting strategy.

Saponin is an important class of natural products with various pharmacological activities. The selective separation of saponins is an essential step before further analysis. Molecular imprinting has been an effective strategy for preparing antibody mimics. However, a facile and efficient imprinting strategy for saponins is still lacking owing to their amphiphilic nature. Herein, we have prepared the saponins imprinted nanoparticles via cooperative imprinting strategy. This new strategy relies on the combination of various non-covalent interactions (hydrophobic and hydrogen bonding) and covalent boronate affinity interactions. The obtained imprinted nanoparticles could rebind specific saponins from complex matrices with good selectivity, superb tolerance to interference, and fast binding equilibrium. This method was verified to be versatile and facile. Thus, this strategy could greatly facilitate the preparation of imprinted nanoparticles for the specific recognition of saponins.

Recent advances of boronate affinity materials in sample preparation.

Boronate affinity materials, as widely used sorbents for the enrichment of cis-diol-containing molecules, have been rapidly developed and increasingly utilized for various applications in recent years. cis-Diol-containing molecules, including saccharides, nucleosides, catecholamines, glycans and glycoproteins/glycopeptides, are major targets in the frontiers of many research areas, such as environmental analysis, the food industry and bioanalysis. As the analysis of these molecules usually suffers from the low abundance of targets and the high abundance of interference, selective enrichment is a fundamental step of sample preparation before analysis. In this review, we survey recent achievements of boronate affinity materials and their applications in sample preparation. We mainly focus on the fundamental considerations of materials as well as important applications in the past 3 years. Particularly, the effects of the substrate structure on the performance of boronate affinity materials, such as binding capacity, affinity, selectivity and working pH, will be discussed. Furthermore, the applications in sample preparation will also be introduced, with a main emphasis on what merits can be provided by boronate affinity materials to overcome the challenges in sample preparation.

Boronate affinity Metal-Organic frameworks for highly efficient cis-diol molecules in-situ enrichment and surface-assisted laser desorption/ionization mass spectrometric detection.

Development of novel matrix for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is a hot-spot in research frontiers. Herein, we established novel SALDI-MS approach for the analysis of cis-diol small molecules by using boronate affinity metal-organic frameworks (BA-MOFs) as the sorbent for extraction and later as matrix for SALDI. In the meantime, the prepared BA-MOFs exhibited several significant merits, such as excellent selectivity, tolerance to interference and fast binding equilibria. By integrating the advantages of MOFs and the molecular recognition of boronate affinity, a novel platform for simultaneous enrichment and MS detection of cis-diol small molecules was proposed for the first time. There are only two steps in the operation procedure, which greatly simplified the whole experimental workflow. Efficient analysis of cis-diol small molecules in real samples were also well demonstrated. This proposed novel protocol not only gave new insights into the application of functionalized MOFs, but also promoted the development of SALDI- MS.

Preparation of salbutamol imprinted magnetic nanoparticles via boronate affinity oriented surface imprinting for the selective analysis of trace salbutamol residues.

Salbutamol (SAL) is one of the most widely abused feed additives in animal husbandry. Selective enrichment of SAL from complex biological samples is a key step for the analysis of SAL. Herein, we present SAL-imprinted magnetic nanoparticles (MNPs) as an ideal sorbent for selective enrichment of SAL. The SAL-imprinted MNPs were prepared according to the newly reported boronate affinity oriented surface imprinting. The prepared imprinted MNPs exhibited several significant advantages, such as excellent selectivity towards SAL, superb tolerance to interferences, fast binding equilibrium and easy manipulation, which made the obtained imprinted MNPs an ideal sorbent for selective enrichment. The feasibility of SAL-imprinted MNPs for real samples was well demonstrated by the analysis of trace SAL in pig liver. Because of their highly desirable properties, the SAL-imprinted MNPs could find more applications in the analysis of SAL.

Efficient synthesis of riboflavin-imprinted magnetic nanoparticles by boronate affinity-based surface imprinting for the selective recognition of riboflavin.

Riboflavin (vitamin B2), a cis-diol-containing compound, is an essential vitamin for maintaining human health mainly in energy metabolism and is a critical component of enzyme cofactors and flavoproteins. Thus, the determination of riboflavin in food products is essential in riboflavin-fortified foods. However, analysis of riboflavin at natural levels in foods or biological samples is difficult because of its very low concentration level and the presence of undesirable matrix effects which could interfere with the measuring system. Thus, it is essential to develop efficient and selective enrichment approaches for riboflavin. Molecularly imprinted polymers can be well used for riboflavin extraction and pre-concentration from food samples. In this study, we present riboflavin-imprinted magnetic nanoparticles as an ideal sorbent for the selective enrichment of riboflavin followed by direct fluorometric determination. The riboflavin-imprinted magnetic nanoparticles were prepared according to a newly reported strategy, called boronate affinity-based surface initiated imprinting. Magnetic nanoparticles and vinylphenylboronic acid were used as supporting materials and a functional monomer, respectively. The prepared riboflavin-imprinted magnetic nanoparticles exhibited several significant advantages, such as excellent selectivity, high binding affinity and low binding pH toward riboflavin, which made the molecularly imprinted material become an ideal sorbent for the selective enrichment of riboflavin. The prepared riboflavin-imprinted magnetic nanoparticles were successfully applied to the analysis of riboflavin in milk.

Precision Imprinting of Glycopeptides for Facile Preparation of Glycan-Specific Artificial Antibodies.

Antibodies specific to glycans are essential in many areas for many important fields, including disease diagnostics, therapeutics, and fundamental researches. However, due to their low immunogenicity and poor availability, glycans pose serious challenges to antibody development. Although molecular imprinting has developed into important methodology for creating antibody mimics with low cost and better stability, glycan-specific molecularly imprinted polymers (MIPs) still remain rather rare. Herein, we report a new strategy, precision imprinting with alternative templates, for the facile preparation of glycan-specific MIPs. Glycopeptides with desirable peptide length immobilized on a boronate affinity substrate were first prepared as alternative templates through in situ dual enzymatic digestion. A thinlayer was then produced to cover the glycans to an appropriate thickness through precision imprinting. With glycoproteins containing only N-glycans as well as both N- and O-glycans as glycan source, this approach was proved to be widely applicable and efficient. The strategy is particularly significant for the recognition of O-glycans, because enzymes that can release O-glycans from O-linked glycoproteins are lacking. The MIPs exhibited excellent glycan specificity. Specific extraction of glycopeptides and glycoproteins containing certain glycans from complex samples was demonstrated. This strategy opened a new avenue for the facile preparation of glycan-specific MIPs, facilitating glycan-related applications and research.

Branched polyethyleneimine-assisted boronic acid-functionalized magnetic nanoparticles for the selective enrichment of trace glycoproteins.

Boronate affinity materials, as efficient sorbents for extraction, separation and enrichment of glycoproteins, have attracted more and more attention in recent years. However, conventional boronate affinity materials suffer from low binding affinity due to the use of single boronic acids, which makes the extraction of glycoproteins in trace concentration rather difficult or impossible. Here we present a type of high boronate avidity material, polyethyleneimine (PEI)-assisted boronic acid-functionalized magnetic nanoparticles (MNPs). Branched PEI was used as a scaffold to amplify the number of boronic acid moieties, while 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA), exhibiting high affinity and excellent water solubility toward cis-diol-containing compounds, was employed as an affinity ligand. Due to the PEI-assisted synergistic multivalent binding, the boronate avidity MNPs exhibited a high binding affinity toward glycoproteins with dissociation constants of 10-6-10-7 M, which was the highest among the reported boronic acid-functionalized materials that can be applied for a glycoproteomic analysis. Such an avidity enabled the selective extraction of trace glycoproteins as low as 2 × 10-15 M. This feature greatly favored the selective enrichment of trace glycoproteins from real samples. Meanwhile, the boronate avidity MNPs were tolerant to the interference of abundant sugars. In addition, the PEI-based boronate avidity MNPs exhibited high binding capacity and low binding pH. The feasibility for practical applications was demonstrated with the selective enrichment of trace glycoproteins in human saliva.

Preparation of molecularly imprinted polymers specific to glycoproteins, glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.

Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited. We developed an approach called boronate affinity controllable-oriented surface imprinting, which allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans and monosaccharides. This approach relies on immobilization of a template (glycoprotein, glycan or monosaccharide) on a boronic-acid-functionalized substrate through boronate affinity interaction, followed by self-polymerization of biocompatible monomer(s) to form an imprinting layer on the substrate with appropriate thickness. Imprinting in this approach is performed in a controllable manner, permitting the thickness of the imprinting layer to be fine-tuned according to the molecular size of the template by adjusting the imprinting time. This not only simplifies the imprinting procedure but also makes the approach widely applicable to a large range of sugar-containing biomolecules. MIPs prepared by this approach exhibit excellent binding properties and can be applied to complex real samples. The MIPs prepared by this protocol have been used in affinity separation, disease diagnosis and bioimaging. The entire protocol, including preparation, property characterization and performance evaluation, takes ∼3-8 d, depending on the type of substrate and template used.

Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers.

Capillary electrophoresis (CE) and magnetic beads have been widely used for the selection of aptamers owing to their efficient separation ability. However, these methods alone are associated with some apparent drawbacks. CE suffers from small injection volumes and thereby only a limited amount of aptamer can be collected at each round. While the magnetic beads approach is often associated with tedious procedure and nonspecific binding. Herein we present a hybrid approach that combines the above two classical aptamer selection methods to overcome the drawbacks associated with these methods alone. In this hybrid method, one single round selection by boronate affinity magnetic nanoparticles (BA-MNPs) was first performed and then followed by a CE selection of a few rounds. The BA-MNPs-based selection eliminated nonbinding sequences, enriching effective sequences in the nucleic acid library. While the CE selection, which was carried out in free solutions, eliminated steric hindrance effects in subsequent selection. Two typical glycoproteins, Ribonuclease B (RNase B) and alkaline phosphatase (ALP), were used as targets. This hybrid method allowed for efficient selection of glycoprotein-binding aptamers within 4 rounds (1 round of BA-MNPs-based selection and 3 rounds of CE selection) and the dissociation constants reached 10-8 M level. The hybrid selection approach exhibited several significant advantages, including speed, affinity, specificity, and avoiding negative selection. Using one of the selected ALP-binding aptamers as an affinity ligand, feasibility for real application of the selected aptamers was demonstrated through constructing an improved enzyme activity assay.

Multimodal Plasmonic Assay of Copper(II) Ion via Stimuli-Responsive State Transformation of Silver Molecular Nanoparticles.

Molecular nanoparticles (MNPs) have gained increased attention recently due to their unique structures and properties. However, their applications remain largely unexplored. Herein, we present an Ag MNPs-based multimodal plasmonic assay. This assay relied on changes in optical properties due to stimuli-responsive state trans-formation from MNPs to plasmonic nanoparticles (PNPs). As a proof-of-concept, naked-eye colorimetric assay, spectrophotometric assay and "turn-on" Raman assay of Cu(2+) were developed. The feasibility of this approach for real-world applications was demonstrated with the determination of Cu(2+) in human serum. This multimodal plasmonic assay exhibited several significant advantages, including selectivity, sensitivity, label-free nature, and multimodal capability. Because of these merits, Ag MNPs could be promising nanosensors for wide important applications such as diagnostics and environmental analysis.