Synthesis of carbon quantum dot-poly lactic-co-glycolic acid hybrid nanoparticles for chemo-photothermal therapy against bacterial biofilms.

Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the resistance to antibiotics. Poly lactic-co-glycolic acid (PLGA)-based nanoparticle delivery systems have been intensively investigated to combat the bacterial biofilms-associated infections. However, some drawbacks associated with current PLGA-based nanoformulations (e.g. the relatively low drug loading capability, premature burst release and/or incapability of on-demand release of cargos at the site of action) restrict the transition from the lab research to the clinical applications. One potent strategy to overcome the above-mentioned limitations is exploiting the unique properties of carbon quantum dots (CQDs) and combining CQDs with the conventional PLGA nanoparticles. In the present study, the CQDs were innovatively incorporated into PLGA nanoparticles by using a microfluidic method. The resulting CQD-PLGA hybrid nanoparticles presented good loading capability of azithromycin (a macrolide antibiotic, AZI) and tobramycin (an aminoglycoside antibiotic, TOB), and stimuli-responsive release of the cargos upon laser irradiation. Consequently, AZI-loaded CQD-PLGA hybrid nanoparticles showed chemo-photothermally synergistic anti-biofilm effects against P. aeruginosa biofilms. Additionally, the CQD-PLGA hybrid nanoparticles demonstrated good biocompatibility with the eukaryotic cells. Overall, the proof-of-concept of CQD-PLGA hybrid nanoparticles may open a new possibility in chemo-photothermal therapy against bacterial biofilms.

Enhanced Plasmon-Induced Resonance Energy Transfer (PIRET)-Mediated Photothermal and Photodynamic Therapy Guided by Photoacoustic and Magnetic Resonance Imaging.

Phototherapy, including photothermal and photodynamic therapy, has attracted extensive attention due to its noninvasive nature, low toxicity, and high anticancer efficiency. The charge-separation mechanism of plasmon-induced resonance energy transfer (PIRET) has been increasingly employed to design nanotheranotic agents. Herein, we developed a novel and smart PIRET-mediated nanoplatform for enhanced, imaging-guided phototherapy. Prussian blue (PB) was incorporated into a Au@Cu2O nanostructure, which was then assembled with poly(allylamine) (PAH)-modified black phosphorus quantum dots (Au@PB@Cu2O@BPQDs/PAH nanocomposites). The hybrid nanosystem exhibited great absorption in the near-infrared region, as well as the ability to self-supply O2 by catalyzing hydrogen peroxide and convert O2 into singlet oxygen (1O2) under 650 nm laser light (0.5 W/cm2) irradiation. In vitro and in vivo assays showed that the generated heat and toxic 1O2 from Au@PB@Cu2O@BPQDs/PAH nanocomposites could effectively kill the cancer cells and suppress tumor growth. Moreover, the unique properties of the PB-modified nanosystem allowed for synergistic therapy with the aid of T1-weighed magnetic resonance imaging (T1-weighted magnetic resonance imaging) and photoacoustic imaging. This study presented a suitable way to fabricate smart PIRET-based nanosystems with enhanced photothermal therapy/photodynamic therapy efficacy and dual-modality imaging functionality. The great biocompatibility and low toxicity ensured their high potential for use in cancer therapy.

Chromatographic separation of hemoglobin variants using robust molecularly imprinted polymers.

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL-1resin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at -2.7 column volumes compared to wildtype HbF.

Fluorometric determination of doxycycline based on the use of carbon quantum dots incorporated into a molecularly imprinted polymer.

A fluorometric assay is described for doxycycline detection. It is based on the use of nitrogen-doped carbon quantum dots (NCQDs) coated with molecularly imprinted polymers (MIPs). The NCQDs were prepared by a one-step hydrothermal reaction using citric acid and ethylenediamine (EDA) as the starting materials. Afterwards, the NCQDs were incorporated into the polymer that was molecularly imprinted with doxycycline. It is found that doxycycline quenches the fluorescence of the NCQDs, and that the functional groups on the surface of NCQDs play an important role in terms of quenching efficiency. A larger fraction of carboxyl groups presented on the surface of NCQDs leads to a higher quenching efficiency due to the enhanced electron transfer from NCQD to doxycycline. The NCQDs@MIP composite can specifically and rapidly recognize doxycycline. Fluorescence drops linearly in the 5 to 50 µM doxycycline concentration range, and the limit of detection is 87 nM. This method was successfully applied to the determination of doxycycline in spiked pig serum where it gave recovery rates of >94%. Graphical abstract Schematic illustration for fabricating a fluorescent sensor based on nitrogen-doped carbon quantum dots (NCQDs) and molecularly imprinted polymers (MIPs). The sensor integrates the merits of the high sensitivity of NCQD and good selectivity of MIPs, and can be significantly quenched upon interaction with doxycycline.

Fluorescent Nanosensor Based on Molecularly Imprinted Polymers Coated on Graphene Quantum Dots for Fast Detection of Antibiotics.

In this work, we developed a novel fluorescent sensor by combining molecularly imprinted polymers (MIPs) with graphene quantum dots (GQDs) for the determination of tetracycline (TC) in aqueous samples. Firstly, we developed a one-pot green method to synthesize GQDs as the fluorescent probes. GQDs with carboxyl groups or amino groups were fabricated. It was found that carboxyl groups played an important role in the fluorescence quenching. Based on these findings, the GQDs-MIPs microspheres were prepared using a sol-gel process. GQDs-MIPs showed strong fluorescent emission at 410 nm when excited at 360 nm, and the fluorescence was quenched in the presence of TC. Under optimum conditions, the fluorescence intensity of GQDs-MIPs decreased in response to the increase of TC concentration. The linear rage was from 1.0 to 104 µg·L-1, and the limit of detection was determined to be 1 µg·L-1. The GQDs-MIPs also demonstrated high selectivity towards TC. The fluorescent sensor was successfully applied for the detection of TC in real spiked milk samples.

Detection of hemoglobin using hybrid molecularly imprinted polymers/carbon quantum dots-based nanobiosensor prepared from surfactant-free Pickering emulsion.

A simple fluorescent nanobiosensor based on molecularly imprinted polymers (MIPs) and carbon quantum dots (CQDs) was developed for hemoglobin (Hb) detection. The nanocomposites were synthesized by a novel one-pot surfactant-free Pickering emulsion method, in which imprinted cavities complementary to Hb were formed at the surface of the particles for target recognition, while CQDs were incorporated in the core as the fluorescence probe. We innovatively used the Hb template as emulsifier to help stabilize the emulsion droplets. The method eliminated the need of surfactant, which greatly simplified Pickering emulsion synthesis procedures, and significantly enhanced the fidelity of molecular imprinting. Moreover, the method provided an easy way to integrate fluorescent probes with MIPs in a single step. The nanobiosensor was utilized for determination of Hb via fluorescence quenching, and high selectivity and sensitivity were achieved. Under the optimized conditions, a linear range of 0.77-7.7 nM and a detection limit of 0.77 nM were obtained. The resulting nanocomposites were also successfully applied to detect Hb in the serum samples, which showed good recoveries ranging from 86.8% to 93.9%.

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction.

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor-made nanomaterials are very promising for use in routine biological assays.

Dispersive solid-phase imprinting of proteins for the production of plastic antibodies.

We describe a novel dispersive solid-phase imprinting technique for the production of nano-sized molecularly imprinted polymers (nanoMIPs) as plastic antibodies. The template was immobilized on in-house synthesized magnetic microspheres instead of conventional glass beads. As a result, high-affinity and template-free MIPs were produced in higher yields.

One-Pot Green Synthesis of Biocompatible Graphene Quantum Dots and Their Cell Uptake Studies.

Graphene-based quantum dots (GQDs) are attractive fluorophores due to their excellent photoluminescence properties, water solubility, low cost, and low toxicity. However, the lack of simple, efficient, and environmental-friendly synthesis methods often limits their biological applications. Herein, we explore a novel, one-pot, green synthesis approach for the fabrication of fluorescent GQDs without involving any harsh reagents. Graphene oxide is used as a precursor, and a 2 h hydrothermal synthesis is carried out with assistance of hydrogen peroxide; no further post purification steps are required. The effects of reaction conditions on the characteristics of GQDs are comprehensively investigated. The as-synthesized GQDs show a high photostability and excellent biocompatibility as revealed by cell viability assays for three different cell lines, namely, macrophages, endothelial cells, and a model cancer cell line. The detailed studies of cellular uptake mechanisms suggest that for all of the three cell lines the major internalization route for GQDs is caveolae-mediated endocytosis followed by clathrin-mediated endocytosis at a less extent. Our results demonstrate the great potential of the as-synthesized GQDs as fluorescent nanoprobes. The study also provides unique insight into the cell-GQDs interactions, which is highly valuable for bioimaging and other related applications such as diagnostics and drug delivery.

Synthesis of naproxen-imprinted polymer using Pickering emulsion polymerization.

For the last decades, molecular imprinting is developing intensively, especially in the case of the application of new imprinting techniques. In this work, for the first time, a Pickering emulsion polymerization was used to synthesize the S-naproxen-imprinted polymer spheres following a noncovalent protocol. To enhance the knowledge about imprinting process using mentioned technique, thorough analysis of the synthesis process was performed. Optimization of polymerization conditions included the selection of functional monomer, cross-linking agent, type of porogen, surfactant, and the choice of appropriate amount of the template and porogen. Prepared materials were characterized using scanning electron microscopy and nitrogen adsorption. To study the binding properties, the sorption studies, including adsorption isotherms and competitive binding, were performed. Investigation of the effect of the functional monomer on the selective recognition of S-naproxen showed that the interactions between the template molecule and 4-vinylpyridine resulted in the best recognizing ability. Moreover, the synthesis with application of ethylene glycol dimethacrylae as a cross-linker, toluene as a porogen, and Tween 20 as an additional emulsion stabilizer gave the most desired result. The optimal ratio of the porogen to monomers mixture was 0.1, due to the fact that the increase of the porogen volume resulted in the significant increase of nonspecific uptake. In addition, the tenfold molar excess of functional monomer relative to the template turned out to be optimal. Subsequent binding studies demonstrated that the material synthesized using optimized polymerization conditions consists of imprinted sites that are sensitive for the S-naproxen.

Preparation of diclofenac-imprinted polymer beads for selective molecular separation in water.

Molecular imprinting technique is an attractive strategy to prepare materials for target recognition and rapid separation. In this work, a new type of diclofenac (DFC)-imprinted polymer beads was synthesized by Pickering emulsion polymerization using 2-(dimethylamino)ethyl methacrylate as the functional monomer. The selectivity and capacity of the molecularly imprinted polymers (MIPs) were investigated in aqueous solution. Equilibrium binding results show that the MIPs have a high selectivity to bind DFC in a wide range of pH values. Moreover, in liquid chromatography experiment, the imprinted polymer beads were packed into column to investigate the binding selectivity under nonequilibrium conditions. The retention time of DFC on the MIP column is significantly longer than its structural analogues. Also, retention of DFC on the MIP column was significantly longer than on the nonimprinted polymer column under aqueous condition. As the new MIP beads can be used to achieve direct separation of DFC from water, the synthetic method and the affinity beads developed in this work opened new possibilities for removing toxic chemicals from environmental and drinking water.

A facile molecularly imprinted polymer-based fluorometric assay for detection of histamine.

Histamine is a biogenic amine naturally present in many body cells. It is also a contaminant that is mostly found in spoiled food. The consumption of foods containing high levels of histamine may lead to an allergy-like food poisoning. Analytical methods that can routinely screen histamine are thus urgently needed. In this paper, we developed a facile and cost-effective molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify histamine. Histamine-specific MIP nanoparticles (nanoMIPs) were synthesized using a modified solid-phase synthesis method. They were then immobilized in the wells of a microplate to bind the histamine in aqueous samples. After binding, o-phthaldialdehyde (OPA) was used to label the bound histamine, which converted the binding events into fluorescent signals. The obtained calibration curve of histamine showed a linear correlation ranging from 1.80 to 44.98 µM with the limit of detection of 1.80 µM. This method was successfully used to detect histamine in spiked diary milk with a recovery rate of more than 85%.

Characterization of Protein-Protein Interactions in Recombinant Hemoglobin Producing Escherichia coli Cells Using Molecularly Imprinted Polymers.

The worldwide blood shortage has generated demands for alternatives to transfusible human blood. One such important option is based on recombinant hemoglobin-based oxygen carriers (rHBOCs). Most efforts have been focused on various E. coli based production systems. One of the key challenges in these systems is to devise an efficient and economical protein production strategy involving selection of suitable host cell and Hb variant, growth conditions and media engineering. Hb also influences the heterologous host cell metabolism and therefore the identification of modified protein-protein interactions is critical for optimizing Hb production. In this study, molecularly imprinted polymers (MIPs) directed against Hb were used to identify the human Hb protein interaction network in E. coli. One E. coli host protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interacted strongly with Hb, especially fetal Hb (HbF).

Nanohybrid polymer brushes on silica for bioseparation.

Boronic acid based affinity materials are of great importance for effective enrichment of biomolecules containing a cis-diol structure, for example glycoproteins. In this work, we developed a new pH- and temperature-responsive boronate affinity material for effective separation of glycoproteins. A nanohybrid material composed of silica cores and flexible polymer brushes, denoted as Si@poly(NIPAm-co-GMA)@APBA, was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) in combination with Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. The size, morphology and composition of the obtained nanohybrid were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), elemental analysis and thermogravimetric analysis (TGA). The density of polymer brushes on the surface of silica nanoparticles was determined to be 0.7 molecules per nm2. The maximum binding capacities of the nanohybrid Si@poly(NIPAm-co-GMA)@APBA for ovalbumin (OVA) and horseradish peroxidase (HRP) were determined to be 87.6 mg g-1 and 22.8 mg g-1, respectively. Glycoprotein binding on the nanohybrid could be controlled by varying the pH of the binding buffer. By increasing the temperature from 20 °C to 35 °C, glycoprotein binding onto the nanohybrid was decreased. This new pH- and temperature-responsive nanohybrid will be useful for a number of biotechnological and biomedical applications, for example, for protein separation and drug delivery.

Implementation of molecularly imprinted polymer beads for surface enhanced Raman detection.

Molecularly imprinted polymers (MIPs) have a predesigned molecular recognition capability that can be used to build robust chemical sensors. MIP-based chemical sensors allow label-free detection and are particularly interesting due to their simple operation. In this work we report the use of thiol-terminated MIP microspheres to construct surfaces for detection of a model organic analyte, nicotine, by surface enhanced Raman scattering (SERS). The nicotine-imprinted microspheres are synthesized by RAFT precipitation polymerization and converted into thiol-terminated microspheres through aminolysis. The thiol groups on the MIP surface allow the microspheres to be immobilized on a gold-coated substrate. Three different strategies are investigated to achieve surface enhanced Raman scattering in the vicinity of the imprinted sites: (1) direct sputtering of gold nanoparticles, (2) immobilization of gold colloids through the MIP's thiol groups, and (3) trapping of the MIP microspheres in a patterned SERS substrate. For the first time we show that large MIP microspheres can be turned into selective SERS surfaces through the three different approaches of assembly. The MIP-based sensing surfaces are used to detect nicotine to demonstrate the proof of concept. As synthesis and surface functionalization of MIP microspheres and nanoparticles are well established, the methods reported in this work are handy and efficient for constructing label-free chemical sensors, in particular for those based on SERS detection.

Preparation of protein imprinted polymer beads by Pickering emulsion polymerization.

We present a new method for preparation of protein-specific polymer beads based on surface molecular imprinting in Pickering emulsion. In the first step, adult human hemoglobin (Hb) was adsorbed on silica nanoparticles. The protein-coated silica particles were then used to stabilize an oil-in-water emulsion (Pickering emulsion) composed of cross-linking monomer in the oil phase. After free radical polymerization of the oil phase, the protein-silica particles were removed to leave Hb-imprinted sites on the polymer surface. The protein-imprinted polymer microspheres were characterized by scanning electron microscopy and their selectivity was investigated by protein binding analysis. The new synthetic method based on Pickering emulsion polymerization produced easily accessible Hb binding sites on the surface of spherical polymer particles, which are useful for protein separation, purification and analysis.

Molecular imprinting of protein in Pickering emulsion.

A new strategy of molecular imprinting to prepare spherical hydrogels via water-in-oil Pickering emulsion polymerization was developed. The imprinted hydrogels exhibited fast adsorption kinetics and significant selectivity for the target protein.