Biomaterial–Related Cell Microenvironment in Tissue Engineering and Regenerative Medicine

An appropriate cell microenvironment is key to tissue engineering and regenerative medicine. Revealing the factors that influence the cell microenvironment is a fundamental research topic in the fields of cell biology, biomaterials, tissue engineering, and regenerative medicine. The cell microenvironment consists of not only its surrounding cells and soluble factors, but also its extracellular matrix (ECM) or nearby external biomaterials in tissue engineering and regeneration. This review focuses on six aspects of biomaterial–related cell microenvironments: ① chemical composition of materials, ② material dimensions and architecture, ③ material–controlled cell geometry, ④ effects of material charges on cells, ⑤ matrix stiffness and biomechanical microenvironment, and ⑥ surface modification of materials. The present challenges in tissue engineering are also mentioned, and eight perspectives are predicted

Lectin- and Saccharide-Functionalized Nano-Chemiresistor Arrays for Detection and Identification of Pathogenic Bacteria Infection

Improvement upon, and expansion of, diagnostic tools for clinical infections have been increasing in recent years. The simplicity and rapidity of techniques are imperative for their adoption and widespread usage at point-of-care. The fabrication and evaluation of such a device is reported in this work. The use of a small bioreceptor array (based on lectin-carbohydrate binding) resulted in a unique response profile, which has the potential to be used for pathogen identification, as demonstrated by Principal Component Analysis (PCA). The performance of the chemiresistive device was tested with Escherichia coli K12, Enterococcus faecalis, Streptococcus mutans, and Salmonella typhi. The limits of detection, based on concanavalin A (conA) lectin as the bioreceptor, are 4.7 × 103 cfu/mL, 25 cfu/mL, 7.4 × 104 cfu/mL, and 6.3 × 102 cfu/mL. This shows that the detection of pathogenic bacteria is achieved with clinically relevant concentrations. Importantly, responses measured in spiked artificial saliva showed minimal matrix interference. Furthermore, the exploitation of the distinctive outer composition of the bacteria and selectivity of lectin-carbohydrate interactions allowed for the discrimination of bacterial infections from viral infections, which is a current and urgent need for diagnosing common clinical infections

DNA Nanostructure Sequence-Dependent Binding of Organophosphates

Understanding the molecular interactions between small molecules and double-stranded DNA has important implications on the design and development of DNA and DNA–protein nanomaterials. Such materials can be assembled into a vast array of 1-, 2-, and 3D structures that contain a range of chemical and physical features where small molecules can bind via intercalation, groove binding, and electrostatics. In this work, we use a series of simulation-guided binding assays and spectroscopy techniques to investigate the binding of selected organophosphtates, methyl parathion, paraoxon, their common enzyme hydrolysis product <i>p</i>-nitrophenol, and double-stranded DNA fragments and DNA DX tiles, a basic building block of DNA-based materials. Docking simulations suggested that the binding strength of each compound was DNA sequence-dependent, with dissociation constants in the micromolar range. Microscale thermophoresis and fluorescence binding assays confirmed sequence-dependent binding and that paraoxon bound to DNA with <i>K</i>_d’s between ∼10 and 300 μM, while methyl parathion bound with <i>K</i>_d’s between ∼10 and 100 μM. <i>p</i>-Nitrophenol also bound to DNA but with affinities up to 650 μM. Changes in biding affinity were due to changes in binding mode as revealed by circular dichroism spectroscopy. Based on these results, two DNA DX tiles were constructed and analyzed, revealing tighter binding to the studied compounds. Taken together, the results presented here add to our fundamental understanding of the molecular interactions of these compounds with biological materials and opens new possibilities in DNA-based sensors, DNA-based matrices for organophosphate extraction, and enzyme–DNA technologies for organophosphate hydrolysis

Enhancing Enzyme Activity and Immobilization in Nanostructured Inorganic–Enzyme Complexes

Understanding the chemical and physical interactions at the interface of protein surfaces and inorganic crystals has important implications in the advancement of immobilized enzyme catalysis. Recently, enzyme–inorganic hybrid complexes have been demonstrated as effective materials for enzyme immobilization. The precipitation of phosphate nanocrystals in the presence of enzymes creates enzyme–Cu₃(PO₄)₂·3H₂O particles with high surface-to-volume ratios, enhanced activity, and increased stability. Here, we begin to develop a mechanistic understanding of enzyme loading in such complexes. Using a series of enzymes including horseradish peroxidase (HRP), a thermostable alcohol dehydrogenase (AdhD), diaphorase, catalase, glucose oxidase (GOx), and the protein bovine serum albumin (BSA), we identified a correlation between particle synthesis temperature, overall enzyme charge, and enzyme loading. The model enzyme HRP has a high predicted pI of ∼7.5 and maintains an overall positive charge under the synthesis conditions, phosphate buffer pH 7.4. HRP loading in HRP–Cu₃(PO₄)₂ complexes was enhanced by 4.2-fold when synthesis was carried out at 37 °C in comparison with synthesis at 4 °C. HRP loading was further enhanced with synthesis at pH 8.0, correlating with a decrease in overall enzyme charge. Proteins with lower predicted pI values and negative overall charge (AdhD, pI of 5.6; diaphorase, pI of 6.8; GOx, pI of 5.2; catalase, pI of 6.9; and, BSA, pI of 5.7) exhibited higher enzyme loadings with 4 °C synthesis, 2.7-, 2.6-, 2.5-, 1.8-, and 1.7-fold protein loading enhancements, respectively. Using HRP as a model system, we also demonstrate that activity increased concomitantly with enzyme loading, and that particle nanostructure was minimally affected by synthesis temperature. Combined, the results presented here demonstrate the control of enzyme loading in enzyme–inorganic particles opening up new possibilities in enzyme and multienzyme catalysis

Electronic Detection of MicroRNA at Attomolar Level with High Specificity

Small RNA (19–23 nucleotides) molecules play an important role in gene regulation, embryonic differentiation, hematopoiesis, and a variety of cancers. Here, we present an ultrasensitive, extremely specific, label-free, and rapid electronic detection of microRNAs (miRNAs) using a carbon nanotubes field-effect transistor functionalized with the Carnation Italian ringspot virus p19 protein biosensor. miRNA-122a was chosen as the target, which was first hybridized to a probe molecule. The probe-miRNA duplex was then quantified by measuring the change in resistance of biosensor resulting from its binding to p19, which selects 21–23 bp RNA duplexes in a size-dependent but sequence-independent manner. The biosensor displayed a wide dynamic range up to 10^–14 M and was able to detect as low as 1 aM miRNA in the presence of a million-fold excess of total RNA, paving the way for simple, point-of-care, low-cost early detection of miRNA as a biomarker in diagnosis of many diseases, including cancer

Tuning Enzyme Kinetics through Designed Intermolecular Interactions Far from the Active Site

Enzyme–DNA nanostructures were designed to introduce new substrate–enzyme interactions into their reactions, which altered enzyme kinetics in a predictable manner. The designed enzymes demonstrate a new strategy of enzyme engineering based on the rational design of intermolecular interactions outside of the active site that enhance and control enzyme kinetics. Binding interactions between tetramethylbenzidine and DNA attached to horseradish peroxidase (HRP) resulted in a reduced Michaelis constant (<i>K</i>_M) for the substrate. The enhancement increased with stronger interactions in the micromolar range, resulting in a 2.6 fold increase in <i>k</i>_cat/<i>K</i>_M. The inhibition effect of 4-nitrobenzoic hydrazide on HRP was also significantly enhanced by tuning the binding to HRP–DNA. Lastly, binding of a nicotinamide adenine dinucleotide (NAD­(H)) cofactor mimic, nicotinamide mononucleotide (NMN­(H)), to an aldo-keto reductase (AdhD) was enhanced by introducing NMN­(H)–DNA interactions