Formation and Structure of Fluorescent Silver Nanoclusters at Interfacial Binding Sites Facilitating Oligomerization of DNA Hairpins.

Fluorescent, DNA-stabilized silver nanoclusters (DNA-AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA-AgNCs with a C-loop hairpin DNA sequence, with subsequent purification by size-exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP-MS), and small-angle X-ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low-resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA-AgNCs self-assemble by a head-to-head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag12 cluster.

Exploiting Femtoliter Microwells for the Sensitive Measurement of Protein Adsorption.

A method is described for the sensitive measurement of adsorbed proteins using femtoliter microwells. Quantitative measurement of adsorbed protein is demonstrated at surface densities from 10 fg/cm2 to 3 pg/cm2. Determination of the efficacy of barrier coatings is also demonstrated using femtoliter microwells. Adsorption at low surface densities is measured, indicating the highest affinity sites on the surface and therefore the initial stages of adsorption. The femtoliter microwell method is shown to be useful in detecting differences between effective protective coatings.

Lipid nanoparticles outperform electroporation in mRNA-based CAR T cell engineering.

Engineered T cells expressing chimeric antigen receptors (CARs) have been proven as efficacious therapies against selected hematological malignancies. However, the approved CAR T cell therapeutics strictly rely on viral transduction, a time- and cost-intensive procedure with possible safety issues. Therefore, the direct transfer of in vitro transcribed CAR-mRNA into T cells is pursued as a promising strategy for CAR T cell engineering. Electroporation (EP) is currently used as mRNA delivery method for the generation of CAR T cells in clinical trials but achieving only poor anti-tumor responses. Here, lipid nanoparticles (LNPs) were examined for ex vivo CAR-mRNA delivery and compared with EP. LNP-CAR T cells showed a significantly prolonged efficacy in vitro in comparison with EP-CAR T cells as a result of extended CAR-mRNA persistence and CAR expression, attributed to a different delivery mechanism with less cytotoxicity and slower CAR T cell proliferation. Moreover, CAR expression and in vitro functionality of mRNA-LNP-derived CAR T cells were comparable to stably transduced CAR T cells but were less exhausted. These results show that LNPs outperform EP and underline the great potential of mRNA-LNP delivery for ex vivo CAR T cell modification as next-generation transient approach for clinical studies.

Preparation of Heat-Denatured Macroaggregated Albumin for Biomedical Applications Using a Microfluidics Platform.

Albumin is widely used in pharmaceutical applications to alter the pharmacokinetic profile, improve efficacy, or decrease the toxicity of active compounds. Various drug delivery systems using albumin have been reported, including microparticles. Macroaggregated albumin (MAA) is one of the more common forms of albumin microparticles, which is predominately used for lung perfusion imaging when labeled with radionuclide technetium-99m (99mTc). These microparticles are formed by heat-denaturing albumin in a bulk solution, making it very challenging to control the size and dispersity of the preparations (coefficient of variation, CV, ∼50%). In this work, we developed an integrated microfluidics platform to create more tunable and precise MAA particles, the so-called microfluidic-MAA (M2A2). The microfluidic chips, prepared using off-stoichiometry thiol-ene chemistry, consist of a flow-focusing region followed by an extended and water-heated curing channel (85 °C). M2A2 particles with diameters between 70 and 300 µm with CVs between 10 and 20% were reliably prepared by adjusting the flow rates of the dispersed and continuous phases. To demonstrate the pharmaceutical utility of M2A2, particles were labeled with indium-111 (111In) and their distribution was assessed in healthy mice using nuclear imaging. 111In-M2A2 behaved similarly to 99mTc-MAA, with lung uptake predominately observed early on followed by clearance over time by the reticuloendothelial and renal systems. Our microfluidic chip represents an elegant and controllable method to prepare albumin microparticles for biomedical applications.

Thiol-Ene Based Polymers as Versatile Materials for Microfluidic Devices for Life Sciences Applications.

While there is a steady growth in the number of microfluidics applications, the search for an optimal material that delivers the diverse characteristics needed for the numerous tasks is still nowhere close to being settled. Often overlooked and still underrepresented, the thiol-ene family of polymer materials has an enormous potential for applications in organs-on-a-chip, droplet productions, microanalytics, and point of care testing. In this review, the main characteristics of the thiol-ene materials are given, and advantages and drawbacks with respect to their potential in microfluidic chip fabrication are critically assessed. Select applications, which exploit the versatility of the thiol-ene polymers, are presented and discussed. It is concluded that, in particular, the rapid prototyping possibility combined with the material's resulting mechanical strength, solvent resistance, and biocompatibility, as well as the inherently easy surface functionalization, are strong factors to make thiol-ene polymers strong contenders for promising future materials for many biological, clinical, and technical lab-on-a-chip applications.

Chloroform compatible, thiol-ene based replica molded micro chemical devices as an alternative to glass microfluidic chips.

Polymeric microfluidic chips offer a number of benefits compared to their glass equivalents, including lower material costs and ease and flexibility of fabrication. However, the main drawback of polymeric materials is often their limited resistance to (organic) solvents. Previously, thiol-ene materials were shown to be more solvent resistant than most other commonly used polymers; however, they still fall short in "harsh" chemical environments, such as when chlorinated solvents are present. Here, we show that a simple yet effective treatment of thiol-ene materials results in exceptional solvent compatibility, even for very challenging chemical environments. Our approach, based on a temperature treatment, results in a 50-fold increase in the chloroform compatibility of thiol-enes (in terms of longevity). We show that prolonged heat exposure allows for the operation of the microfluidic chips in chloroform for several days with no discernable deformation or solvent-induced swelling. The method is applicable to many different thiol-ene-based materials, including commercially available formulations, and also when using other commonly considered "harsh" solvents. To demonstrate the utility of the solvent compatible thiol-enes for applications where chloroform is frequently employed, we show the continuous and uniform production of polymeric microspheres for drug delivery purposes over a period of 8 hours. The material thus holds great promise as an alternative choice for microfluidic applications requiring harsh chemical environments, a domain so far mainly restricted to glass chips.

The structural shift of a DNA template between a hairpin and a dimer tunes the emission color of DNA-templated AgNCs.

The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.