Probing Protein 4'-Phosphopantetheinylation in Single Living Cells.

4'-Phosphopantetheinylation (4PPTylation) of proteins, which is derived from the hydrolysis of coenzyme A (CoA), is an essential post-translational modification participating in biosynthetic and metabolic pathways. However, due to the lack of specific recognition ligands as well as the shortage of sensitive analytical tools for single-cell analysis, the in-depth exploration of new cellular functions and mechanisms of protein 4PPTylation has been much hampered. In this study, we rationally engineered CoA-imprinted Raman nanotags for the specific recognition of 4PPTylation and thereby developed a molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA) for facile probing the 4PPTylation of ALDH1L1 in single cells. The molecularly imprinted nanotags exhibited excellent binding properties, giving a dissociation constant of 10-6 M and cross-reactivity values of less than 10%. The MIP-based PISA enabled the specific and sensitive detection of the level of 4PPTylated ALDH1L1 in single living cells. Particularly, monitoring of the fluctuation of 4PPTylated ALDH1L1 in single cells under simulation by an inhibitor (methotrexate) that acts on a different metabolism pathway was achieved, implying possible crosstalk between two different pathways in folate metabolism. Thus, the imprinted Raman nanotags-PISA provides a promising analytical tool with a single-cell resolution for exploring new functions and elucidating their mechanisms of protein 4PPTylation.

Molecularly Imprinted Nanozymes with Free Substrate Access for Catalyzing the Ligation of ssDNA Sequences.

Nanozymes have attracted wide attention for the unique advantages of low cost, high stability and designability. Molecularly imprinted polymers (MIPs) have demonstrated great potential as a new type of nanozymes due to their excellent specificity and high affinity. However, effective approaches for creating molecularly imprinted nanozymes still remain limited. Herein, reverse microemulsion template docking surface imprinting (RMTD-SI) is reported as a new approach for the rational design and engineering of nanozymes with free substrate access for the ligation of ssDNA sequences. As a proof of the principle, octa-deoxyribonucleotide-imprinted nanoparticles were successfully prepared. Using tetradeoxyribonucleotides and octa-deoxyribonucleotide as substrates, the properties, catalytic activity and behavior of the imprinted nanoparticles were thoroughly investigated. The imprinted nanozyme exhibited an enhanced reaction speed (by up to 41-fold) and good sequence selectivity towards substrate tetra-deoxyribonucleotides. More interestingly, due to the open substrate access, the imprinted nanozyme also allowed the ligation of a ssDNA that fully matched with the imprinted cavity and other ssDNA substrates to form longer sequences, but at the price of substrate selectivity. Thus, this study provides not only a new avenue to the rational design and synthesis of molecularly imprinted nanozymes but also new insights of their catalytic behavior.

Molecularly imprinted and cladded nanoparticles for high-affinity recognition of structurally closed gangliosides.

A new method called reverse microemulsion-confined ganglioside-oriented surface imprinting and cladding (RM-GOSIC) is presented for controllable preparation of nanoscale binders for high-affinity targeting gangliosides. Using GM1a, an affordable ganglioside, as a representative ganglioside target, single-core quantum dot GM1a-imprinted and GM1a-cladded polymer (cMIP) nanoparticles were prepared. The prepared cMIP nanoparticles exhibited extremely high affinity towards GM1a, with dissociation constant at the nanomolar level (3-6 nM). The prepared cMIP nanoparticles also recognized structurally closed gangliosides while their cross-reactivity towards other gangliosides remained low. The potential of the cMIP nanoparticles in biomedical applications was demonstrated by cell and tissue imaging. Thus, this approach opened a new access to the synthesis of high-affinity nanoscale binders for targeting gangliosides.

Controllably Prepared Aptamer-Molecularly Imprinted Polymer Hybrid for High-Specificity and High-Affinity Recognition of Target Proteins.

Molecularly imprinted polymers (MIPs) and aptamers, as effective mimics of antibodies, can overcome only some drawbacks of antibodies. Since they have their own advantages and disadvantages, the combination of MIPs with aptamers could be an ideal solution to produce hybrid alternatives with improved properties and desirable features. Although quite a few attempts have been made in this direction, a facile and controllable approach for the preparation of aptamer-MIP hybrids still remains lacking. Herein, we present a new approach for facile and controllable preparation of aptamer-MIP hybrids for high-specificity and high-affinity recognition toward proteins. An aptamer that can bind the glycoprotein alkaline phosphatase (ALP) with relative weak affinity and specificity was used as a ligand, and controllable oriented surface imprinting was carried out with an in-water self-polymerization system of dopamine. A thin layer of polydopamine was formed to cover the template to an appropriate thickness. After removing the template from the polymer, an aptamer-MIP hybrid with apparently improved affinity and specificity toward ALP was obtained, giving cross-reactivity of 3.2-5.6% and a dissociation constant of 1.5 nM. With this aptamer-MIP hybrid, a plasmonic immunosandwich assay (PISA) was developed. Reliable detection of ALP in human serum by the PISA was demonstrated.

Molecularly imprinted and cladded polymers for constructing a portable plasmonic immunoassay for peptides in biofluids.

Using two molecularly imprinted and cladded polymers (cMIPs), an inexpensive, fast and portable plasmonic immuno-sandwich assay (PISA) was rationally developed for high-specificity and ultra-sensitive detection of C-peptide in urine. The dual cMIPs-based PISA allowed healthy individuals to be distinguished from diabetes patients and exhibited several significant merits over existing immunoassays, holding great promise in clinical diagnosis.

Construction of DNA ligase-mimicking nanozymes via molecular imprinting.

Enzyme mimics are of significant importance due to their facile preparation, low cost and stability to rigorous environments. Molecularly imprinted polymers (MIPs) have been important synthetic mimics of enzymes. However, effective strategies for the rational design of enzyme-mimicking MIPs have still remained limited. Herein, we report a new strategy, termed affinity gathering-enhanced coupling and thermal cycling amplification (AGEC-TCA), for the rational design and engineering of molecularly imprinted mesoporous silica nanoparticles (MSNs) that are capable of ligating short ssDNA fragments. This strategy relied on enhancing the effective collision probability via binding substrates into highly favorable orientation by product-imprinted MSNs as well as product release via thermal cycling which enabled successive product amplification. Using modified and natural hexadeoxyribonucleotide as templates, the prepared product-imprinted MSNs exhibited a remarkably enhanced reaction speed (by up to 63-fold) as well as excellent sequence specificity towards substrate trideoxyribonucleotides. Thus, this strategy opened up a new avenue to access enzyme mimics via molecular imprinting.

Controllably prepared molecularly imprinted core-shell plasmonic nanostructure for plasmon-enhanced fluorescence assay.

Plasmon-enhanced fluorescence (PEF) is an emerging technology for sensitive detection. It relies on the plasmonic effect of a noble metal nanostructure to dramatically enhance the fluorescence of target fluorophores around the metal surface. Because there is a compromise between plasmonic enhancement and fluorescence quenching, it is critical to control the distance between the fluorophore and the metal surface to an appropriate range. This makes the fabrication of plasmonic nanostructures for PEF assays a challenging task. Herein, we report a controllably prepared core-shell plasmonic nanostructure coated with molecularly imprinted polymer (MIP) for sensitive and specific PEF assay. Riboflavin (RF) was used as a test compound in this study. RF-imprinted Ag@SiO2 nanoparticles were prepared in a controllable manner, providing an optimal distance between the metal surface and RF molecules. The obtained hybrid nanostructure allowed for sensitive detection and specific recognition towards the target. Based on the plasmonic hybrid nanostructure, a sensitive and specific PEF assay of RF was developed and successfully applied to the determination of RF in human urine. Thus, the study paved the way for controllable preparation of molecularly imprinted plasmonic nanostructures for sensitive and specific PEF assays.

Adsorption of Rh(III) complexes from chloride solutions obtained by leaching chlorinated spent automotive catalysts on ion-exchange resin Diaion WA21J.

It was found that Rh, Pd and Pt contained in the spent ceramic automotive catalysts could be effectively extracted by dry chlorination with chlorine. In order to concentrate Rh(III) ions contained in the chloride solutions obtained, thermodynamic and kinetics studies for adsorption of Rh(III) complexes from the chloride solutions on an anionic exchange resin Diaion WA21J were carried out. Rh, Pd, Pt, Al, Fe, Si, Zn and Pb from the chloride solution could be adsorbed on the resin. The distribution coefficients (K(d)) of Rh(III) decreased with the increase in initial Rh(III) concentration or in adsorption temperature. The isothermal adsorption of Rh(III) was found to fit Langmuir, Freundlich and Dubinin-Kaganer-Radushkevich models under the adsorption conditions. The maximum monolayer adsorption capacities Q(max) based on Langmuir adsorption isotherms were 6.39, 6.61 and 5.81 mg/g for temperatures 18, 28 and 40 degrees C, respectively. The apparent adsorption energy of Rh was about -7.6 kJ/mol and thus Rh(III) adsorption was a physical type. The experimental data obtained could be better simulated by pseudo-first-order kinetic model and the activation energy obtained was 6.54 J/mol. The adsorption rate of Rh(III) was controlled by intraparticle diffusion in most of time of adsorption process.