Molecular Immobilization Research Articles

Synthesis of isocyanate containing cryogels for fast (bio)molecular immobilization.

Cryogels containing isocyanate reactive groups were synthesized via photopolymerization of 2-isocyanoethyl methacrylate (ICEMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA). By changing the PEGMEMA and ICEMA ratios, cryogel series with varying ratios of reactive isocyanate groups were successfully prepared. The cryogels were characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis, scanning electron microscope, and rheometry. To demonstrate that molecules containing amine groups can be immobilized onto synthesized cryogels through isocyanate-amine reactions, the cryogels were conjugated with 4-(trifluoromethyl)benzylamine (TFBA) and fluorescein amine (FLA) molecules, and the conjugations were confirmed through FTIR and fluorescence microscopy, respectively, for TFBA and FLA. Additionally, immobilization of fluorescein isothiocyanate conjugated albumin from bovine serum (FITC-BSA) as fluorescein-labeled model proteins was studied to illustrate that biomolecules can also be bound to the cryogels without any linker. It was shown that the amount of immobilized FITC-labeled model proteins can be controlled by adjusting the concentration of isocyanate reactive groups within the cryogel matrix, and this functionalization was confirmed by fluorescence microscope.

Targeted recognition and enhanced biotransformation of phytochemicals by a dual-functional cellulose-based hydrogel bioreactor

The combined technology of molecular imprinting and immobilization is a promising strategy for improving biocatalytic performance. In this work, a dual-functional cellulose-based hydrogel bioreactor with targeted recognition and transformation functions was innovatively designed by cellulose-based molecularly imprinted polymers (CM-MIPs) hydrogel coupled with layered double hydroxide immobilized enzyme (LDH-E). Firstly, CM-MIPs hydrogel was prepared by using phlorizin, 4-pinylpyridine, and cellulose microspheres as template molecule, functional monomer, and support material, respectively. Meanwhile, layered double hydroxide (LDH) taken as a carrier to immobilize β-glucosidase. The prepared LDH was layered sheet-like structure with ultra-thin thickness of approximately only 1.5 nm, and β-glucosidase was immobilized on both sides of the LDH. Further, the dual-functional bioreactor was constructed by the anion exchange, which possessed maximum targeted adsorption capacity to phlorizin of 15.25 mg/g, exhibited 1.2 folds higher transformation efficiency than LDH-E, and the phloretin content increased 26 folds to that in Lithocarpus litseifolius (Hance) Chu leaves extracts. Moreover, the transformation efficiency remained above 70 % even after five consecutive transformations. Overall, the dual-functional bioreactor has broad prospects for the application in targeted recognition and transformation of phytochemicals, and provides a new insight on multifunctional bio-based reactors in natural production field.

Design and Synthesis of Heterogeneous Frustrated Lewis Pairs for Hydrogenation: From Molecular Immobilization to Defects Engineering

AbstractThe concept of “Frustrated Lewis pairs” (FLPs), which emerged from the discovery that H2 can be reversibly activated by combinations of sterically encumbered Lewis acids and bases. Since then, the field of FLP chemistry has expanded enormously. One of the most remarkable achievements is the development of FLP catalysts for hydrogenation, which are environmentally friendly and have potential industrial application prospects. In recent years, this unique catalysis concept has pushed the study of FLP chemistry to a new direction: heterogeneous FLP catalysts. This review outlines the recent research progress of new strategies to design and synthesize heterogeneous FLPs for hydrogenation reaction, and prospects the development of novel heterogeneous FLP catalysts.

A Cascade Strategy Boosting Hydroxyl Radical Generation with Aggregation-Induced Emission Photosensitizers-Albumin Complex for Photodynamic Therapy.

Effective photodynamic therapy (PDT) requires photosensitizers (PSs) to massively generate type I reactive oxygen species (ROS) in a less oxygen-dependent manner in the hypoxia tumor microenvironment. Herein, we present a cascade strategy to boost type I ROS, especially hydroxyl radical (OH·-), generation with an aggregation-induced emission (AIE) photosensitizer-albumin complex for hypoxia-tolerant PDT. The cationic AIE PS TPAQ-Py-PF6 (TPA = triphenylamine, Q = anthraquinone, Py = pyridine) contains three important moieties to cooperatively enhance free radical generation: the AIE-active TPA unit ensures the effective triplet exciton generation in aggregate, the anthraquinone moiety possesses the redox cycling ability to promote electron transfer, while the cationic methylpyridinium cation further increases intramolecular charge transfer and electron separation processes. Inserting the cationic TPAQ-Py-PF6 into the hydrophobic domain of bovine serum albumin nanoparticles (BSA NPs) could greatly immobilize its molecular geometry to further increase triplet exciton generation, while the electron-rich microenvironment of BSA ultimately leads to OH·- generation. Both experimental and theoretical results confirm the effectiveness of our molecular cationization and BSA immobilization cascade strategy for enhancing OH·- generation. In vitro and in vivo experiments validate the excellent antitumor PDT performance of BSA NPs, superior to the conventional polymeric encapsulation approach. Such a multidimensional cascade strategy for specially boosting OH·- generation shall hold great potential in hypoxia-tolerant PDT and related antitumor applications.

Monitoring levamisole in food and the environment with high selectivity using an electrochemical chiral sensor comprising an MOF and molecularly imprinted polymer

We used an enhanced recognition strategy to fabricate a novel levamisole-detecting chiral electrochemical sensor featuring a metal–organic framework (MOF) combined with a molecularly imprinted polymer (MIP). We first synthesised a Cu/Zn-[benzene-1,3,5-tricarboxylic acid] (Cu/Zn-BTC) MOF as the molecular immobilisation and signal-amplifying unit, and then prepared the MIP (molecular recognition unit) using levamisole as the template on a glassy carbon electrode modified with Cu/Zn-BTC. We obtained a composite chiral sensor with enhanced recognition capability for levamisole after template removal. Using the templated sites as the switch and K3[Fe(CN)6]/K4[Fe(CN)6] as a probe, we established a new method for detecting levamisole in meat products and water bodies. The linear detection range and detection limit of our chiral sensor are 5 to 6000 × 10-11 mol/L and 1.65 × 10−12 mol/L, respectively. Moreover, the sensor exhibited 93.8–109.0% recovery in the detection of levamisole in chicken and other real samples.

Enzymatic Fabrication of Multifunctional Amyloid-based Protein Assembly

We developed a method to orthogonally immobilize multiple functional molecules on amyloid aggregates using different enzymes (sortase A and transglutaminase). Orthogonal binding was performed via the substrate amino acid sequence of each enzyme, and the amount of molecular immobilization could be changed according to the amount of substrate sequence. This method is expected to have applications such as improving the sensitivity of immunoassays and visualization of amyloid elongation reactions. We developed a method for the enzymatic introduction of two types of functional molecules onto amyloids. Immobilization of the functional molecules was conducted via the substrate sequence, and no loss of function of the molecules was observed. Our method enables the installation of molecules with suitable substrate sequences, thereby providing a means to create complexes with diverse functions.

Biosensors Based on the Binding Events of Nitrilotriacetic Acid-Metal Complexes.

Molecular immobilization and recognition are two key events for the development of biosensors. The general ways for the immobilization and recognition of biomolecules include covalent coupling reactions and non-covalent interactions of antigen-antibody, aptamer-target, glycan-lectin, avidin-biotin and boronic acid-diol. Tetradentate nitrilotriacetic acid (NTA) is one of the most common commercial ligands for chelating metal ions. The NTA-metal complexes show high and specific affinity toward hexahistidine tags. Such metal complexes have been widely utilized in protein separation and immobilization for diagnostic applications since most of commercialized proteins have been integrated with hexahistidine tags by synthetic or recombinant techniques. This review focused on the development of biosensors with NTA-metal complexes as the binding units, mainly including surface plasmon resonance, electrochemistry, fluorescence, colorimetry, surface-enhanced Raman scattering spectroscopy, chemiluminescence and so on.

Plier Ligands for Trapping Neurotransmitters into Complexes for Sensitive Analysis by SERS Spectroscopy

Catecholamines-dopamine, noradrenaline and adrenaline are important biomarkers of neurotransmitter metabolism, indicating neuroendocrine tumors and neurodegenerative diseases. Surface-enhanced Raman spectroscopy (SERS) is a promising analytical technique with unprecedented multiplexing capabilities. However, not all important analytes exhibit strong SERS signals on stable and robust nanostructured substrates. In this work, we propose a novel indicator system based on the formation of mixed ligand complexes with bispidine-based bis-azole ligands which can serve as pliers to trap Cu(II) ions and stabilize its complexes with catecholamines. Four synthesized ligands with different functional groups: carboxyl, amino, benzyl, and methoxybenzyl, were applied for forming stable complexes to shift maximum absorbance of catecholamines from the ultraviolet region to 570-600 nm. A new absorbance band in the visible range resonates with the local surface plasmon resonance (LSPR) band of metal nanoparticles and most used laser wavelengths. This match allowed use of Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) methodology to measure intense Raman signals on a nanostructured silver-based SERS-active substrate. The synthesized plier-like ligands fixed and stabilized catecholamine complexes with Cu(II) on the SERS sensor surface, which facilitated the determination of dopamine in a 3.2 × 10-12-1 × 10-8 M concentration range.

Microbial elimination of pyrethroids: specific strains and involved enzymes.

Pyrethroids, which are synthetic organic insecticides, are widely used in agriculture and households to resist pests and control disease transmission. However, pyrethroids have inevitably caused environmental pollution, leading to concerns for food safety and human health. Bioremediation has emerged as one of the most promising methods to eliminate pyrethroids compounds. Pyrethroid-degrading microorganisms and the relevant enzymes have shown an efficient ability in degrading pyrethroids by hydrolyzing the ester linkage. In this review, a wide variety of pyrethroid-degrading strains were presented and classified from different sources, such as wastewater, soils, and oceans. In addition, the recombinant expression, enzyme identification, and molecular modification of these microbial pyrethroid-degrading enzymes were also compared and discussed in detail. Moreover, the potential applications of pyrethroid-degrading enzymes, including immobilization and biodegradation towards a series of pyrethroids, were also presented. All of the positive results obtained from this review could be a good guideline for the other research in this field. KEY POINTS: • Distribution of pyrethroid-degrading strains in different sources was summarized. • Enzymatic properties including pH, temperature, and substrate specificity were compared. • Promising molecular modification and immobilization of hydrolases were present.

Molecular Engineering of Metal Complexes for Electrocatalytic Carbon Dioxide Reduction: From Adjustment of Intrinsic Activity to Molecular Immobilization.

The electrocatalytic CO2 reduction reaction (ECO2 RR) is one promising method for storing intermittent clean energy in chemical bonds and producing fuels. Among various kinds of catalysts for ECO2 RR, molecular metal complexes with well-defined structures are convenient for studies of their rational design, structure-reactivity relationships, and mechanisms. In this Review, we summarize the molecular engineering of several N-based metal complexes including Re/Mn bipyridine compounds and metal macrocycles, concluding with general modification strategies to devise novel molecular catalysts with high intrinsic activity. Through physical adsorption, covalent linking, and formation of a periodic backbone, these active molecules can be heterogenized into immobilized catalysts with more practical prospects. Finally, significant challenges and opportunities based on molecular catalysts are discussed.

Molecular Engineering of Metal Complexes for Electrocatalytic Carbon Dioxide Reduction: From Adjustment of Intrinsic Activity to Molecular Immobilization

AbstractThe electrocatalytic CO2 reduction reaction (ECO2RR) is one promising method for storing intermittent clean energy in chemical bonds and producing fuels. Among various kinds of catalysts for ECO2RR, molecular metal complexes with well‐defined structures are convenient for studies of their rational design, structure–reactivity relationships, and mechanisms. In this Review, we summarize the molecular engineering of several N‐based metal complexes including Re/Mn bipyridine compounds and metal macrocycles, concluding with general modification strategies to devise novel molecular catalysts with high intrinsic activity. Through physical adsorption, covalent linking, and formation of a periodic backbone, these active molecules can be heterogenized into immobilized catalysts with more practical prospects. Finally, significant challenges and opportunities based on molecular catalysts are discussed.

Hemocompatibility Evaluation of Thai Bombyx mori Silk Fibroin and Its Improvement with Low Molecular Weight Heparin Immobilization.

Bombyx mori silk fibroin (SF), from Nangnoi Srisaket 1 Thai strain, has shown potential for various biomedical applications such as wound dressing, a vascular patch, bone substitutes, and controlled release systems. The hemocompatibility of this SF is one of the important characteristics that have impacts on such applications. In this study, the hemocompatibility of Thai SF was investigated and its improvement by low molecular weight heparin (LMWH) immobilization was demonstrated. Endothelial cell proliferation on the SF and LMWH immobilized SF (Hep/SF) samples with or without fibroblast growth factor-2 (FGF-2) was also evaluated. According to hemocompatibility evaluation, Thai SF did not accelerate clotting time, excess stimulate complement and leukocyte activation, and was considered a non-hemolysis material compared to the negative control PTFE sheet. Platelet adhesion of SF film was comparable to that of the PTFE sheet. For hemocompatibility enhancement, LMWH was immobilized successfully and could improve the surface hydrophilicity of SF films. The Hep/SF films demonstrated prolonged clotting time and slightly lower complement and leukocyte activation. However, the Hep/SF films could not suppress platelet adhesion. The Hep/SF films demonstrated endothelial cell proliferation enhancement, particularly with FGF-2 addition. This study provides fundamental information for the further development of Thai SF as a hemocompatible biomaterial.

Evaluating glioma-associated microRNA by complementation on a biological nanosensor.

Glioma is a tumor in the brain and spinal cord originating in the glial cells that surround the nerve cells. Among several microRNAs reported, miRNA-363 is associated with human glioma. Based on miRNA-363 levels, the development and progression of glioma can be monitored. The current study used an interdigitated electrode sensor to monitor microRNA-363 levels, which indeed reflects the severity of glioma. The interdigitated electrode was generated using a photolithography technique followed by surface chemical modification carried out to insert miRNA-363 complementary oligo as the probe complexed with gold nanoparticles. The proposed sensor works based on the dipole moment between two electrodes, and when molecular immobilization or interaction occurs, the response by the signal output changes. The changes in the target microRNA-363 sequence were standardized to identify glioma. The limit of detection of miRNA-363 was 10 fM with an R2 value of 0.996 on the linear coefficient regression ranges between 1 fM and 100 pM. Furthermore, unrelated sequences failed to increase the response of the current with the complementary probe, indicating specific miRNA-363 detection on the interdigitated electrode. This study demonstrates the platform to be used for determining the presence of microRNA-363 in glioma and as the basis for other biomarker analyses.

Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates for multiplex determination of dopamine, norepinephrine, and epinephrine.

Aunique approach based on Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates is proposed for sensitive and rapid determination of the catecholamines (CA)dopamine, norepinephrine, and epinephrine. The ternary (CA)2Cu(4AAP)2 complexes were characterized by the appearance of new absorbance bands at 555, 600, and 500nm for dopamine, norepinephrine, and epinephrine, respectively. The new absorbance band matched with a broad surface plasmon resonance band of utilized silver nanoparticles: 450-600nm, and 633excitation wavelength. We observed enhancement factors up to 3.6·106 due to the additional resonant enhancement. The multiplexing capabilities of quantitative spectral unmixing for Raman spectra of a group of CAs, which differ by only either hydroxy or methyl group, at the fingerprint region were successfully demonstrated with the direct classic least squares model. The achieved nM limits of detection with only 1.5 mW laser power and analysis of spiked human blood plasma samples proved the possibility of the multiplex determination of the catecholamines at the level of reference concentrations in the blood of healthy people as well as promise for the future facilitation in the precision diagnosis of neuroendocrine tumors and neurodegenerative diseases.

Evaluation of Dibenzocyclooctyne and Bicyclononyne Click Reaction on Azido‐Functionalized Antifouling Polymer Brushes via Microspotting

AbstractStrain‐promoted alkyne‐azide cycloaddition (SPAAC) has become an indispensable tool in bioorthogonal conjugation and surface immobilization. While numerous studies have focused on enhancing the reactivity of cyclooctynes, a facile method to evaluate the binding efficiency for cyclooctyne‐azide‐based immobilization without any sophisticated facilities is still missing. In the present work, different derivatives of dibenzocyclooctyne/bicyclononyne (DBCO/BCN) linked to either a fluorophore or a biotin‐moiety are patterned on ultra‐low fouling polymer brushes, which can avoid unspecific protein contamination without any prior blocking steps. The polymer brushes are composed of an antifouling bottom block and azide‐terminated top block. The assessment of binding efficiency is conducted on ordered arrays spotted by microchannel cantilever spotting (μCS) with a normal fluorescent microscope. Both cyclooctynes demonstrate reliable binding performance with azide‐bearing diblock polymer brushes via μCS, but DBCO shows a higher surface density of molecular immobilization according to the protein binding assays. This work provides a reference for choosing appropriate cyclooctyne to couple with azides and can be useful for the design of biosensors or bio‐platforms for analyte detection, cell capture, and other biological applications.

Ultrasensitive and multiplex SERS determination of anthropogenic phenols in oil fuel and environmental samples

SERS with molecular immobilization and resonant Raman amplification by complex-loaded enhancer (MIRRACLE) enables detection of hazardous phenolic pollutants down to nM concentrations in gasoline fuel and freshwater samples.

Rapid Detection of SARS-CoV-2 Antigens and Antibodies Using OFET Biosensors Based on a Soft and Stretchable Semiconducting Polymer.

In the midst of the COVID-19 pandemic, adaptive solutions are needed to allow us to make fast decisions and take effective sanitation measures, e.g., the fast screening of large groups (employees, passengers, pupils, etc.). Although being reliable, most of the existing SARS-CoV-2 detection methods cannot be integrated into garments to be used on demand. Here, we report an organic field-effect transistor (OFET)-based biosensing device detecting of both SARS-CoV-2 antigens and anti-SARS-CoV-2 antibodies in less than 20 min. The biosensor was produced by functionalizing an intrinsically stretchable and semiconducting triblock copolymer (TBC) film either with the anti-S1 protein antibodies (S1 Abs) or receptor-binding domain (RBD) of the S1 protein, targeting CoV-2-specific RBDs and anti-S1 Abs, respectively. The obtained sensing platform is easy to realize due to the straightforward fabrication of the TBC film and the utilization of the reliable physical adsorption technique for the molecular immobilization. The device demonstrates a high sensitivity of about 19%/dec and a limit of detection (LOD) of 0.36 fg/mL for anti-SARS-Cov-2 antibodies and, at the same time, a sensitivity of 32%/dec and a LOD of 76.61 pg/mL for the virus antigen detection. The TBC used as active layer is soft, has a low modulus of 24 MPa, and can be stretched up to 90% with no crack formation of the film. The TBC is compatible with roll-to-roll printing, potentially enabling the fabrication of low-cost wearable or on-skin diagnostic platforms aiming at point-of-care concepts.

Molecular engineering of organic-based agents for in situ bioimaging and phototherapeutics.

In situ monitoring of the location and transportation of bioactive molecules is essential for deciphering diverse biological events in the field of biomedicine. In addition, obtaining the in situ information of lesions will provide a clear perspective for surgeons to perform precise resection in clinical surgery. Notably, delivering drugs or operating photodynamic therapy/photothermal therapy in situ by labeling the lesion regions of interest can improve treatment and reduce side effects in vivo. In various advanced imaging and therapy modalities, optical theranostic agents based on organic small molecules can be conveniently modified as needed and can be easily internalized into cells/lesions in a non-invasive manner, which are prerequisites for in situ bioimaging and precision treatment. In this tutorial review, we first summarize the in situ molecular immobilization strategies to retain small-molecule agents inside cells/lesions to prevent their diffusion in living organisms. Emphasis will be focused on introducing the application of these strategies for in situ imaging of biomolecules and precision treatment, particularly pertaining to why targeting therapy in situ is required.

The preparation of felodipine/zein amorphous solid dispersions and in vitro evaluation using a dynamic gastrointestinal system

ABSTRCT Felodipine has been widely used as a poorly water-soluble model drug for various studies to improve its oral bioavailability and in vivo efficacy. In this study, we developed amorphous solid dispersions (ASDs) via spray drying to enhance the bioavailability of felodipine through using natural zein protein as a novel polymeric excipient. The solid state characterization results demonstrated a single glass transition temperature (Tg ) around 128.6 °C and good physical stability post 3 months accelerated study under the condition of 40 °C and 75% relative humidity (RH), which is possibly accounted for the molecular immobilization and hydrogen bonding interactions between felodipine and zein. By combining the in vitro dissolution study with TIM-1 gastrointestinal simulation investigation, it is indicated that felodipine was rapidly released from the ASD in 30 mins, and the supersaturation of felodipine was well maintained over 6 h, which resulted in a significant enhancement of felodipine bioavailability during simulated digestive processes in the upper GI tract. This study suggests that spray drying combined with natural excipient zein is an efficient formulation strategy for the development of ASDs with enhanced aqueous solubility and bioavailability.

Advances in Organic Transistor‐Based Biosensors

AbstractOrganic transistors are promising candidates for various biosensing applications owing to their flexibility, biocompatibility, and large‐scale processability. Until now, the construction of state‐of‐the‐art biosensors based on organic transistors, which relies on the fine‐tuned interaction between active layers and target analytes, is highly desired but remains challenging. Herein, the key strategies for constructing sensitive and selective biosensors based on organic transistors via device engineering are summarized. Recent achievements, including the molecular design of organic semiconductors, bio‐receptors assembly through the molecular immobilization, and strategies for overcoming the Debye screening effect, are discussed in a focused manner.