Microfluidic electrochemical biosensors: tools for advancing the sustainable development goals.

New technologies can help to achieve the sustainable development goals (SDGs) of the United Nations. We discuss the contribution of microfluidic electrochemical biosensors to advancing the SDGs. These sensors can be applied in various fields given their low cost, self-powering ability, environmental compatibility, ease of use, and small sample volume requirements.

Antimicrobial Peptide Assembly on Zwitterionic Polymer Films to Slow Down Biofilm Formation.

Formation of biofilms on equipment used in various fields, such as medicine, domestic sanitation, and marine transportation, can cause serious problems. The use of antibiofouling and bactericidal modifications is a promising strategy for inhibiting bacterial adhesion and biofilm formation. To further enhance the antibiofilm properties of a surface, various combinations of bactericidal modifications alongside antibiofouling modifications have been developed. Optimization of the arrangements of antimicrobial peptides on the antibiofouling surface would allow us to design longer-life antibiofilm surface modifications. In this study, a postmodification was conducted with different design using the antimicrobial peptide KR12 on an antibiofouling copolymer film consisting of 2-methacryloyloxyethyl phosphorylcholine, 3-methacryloxypropyl trimethoxysilane, and 3-(methacryloyloxy) propyl-tris(trimethylsilyloxy) silane. The distance of KR12 from the film was adjusted by combining different lengths of poly(ethylene glycol) (PEG) spacers (molecular weights are 2000 and 5000). The density of KR12 was ranged from 0.06 to 0.22 nm-2. When these modified surfaces were exposed to a nutrient-rich TSB suspension, the bacterial area formed by E. coli covered 5-127% of the original copolymer film. We found that a significant distance between the bactericidal and antibiofouling modifications, along with a higher density of bactericidal modifications, slows down the biofilm formation.

Superior antibacterial surfaces using hydrophilic, poly(MPC) and poly(mOEGMA) free chains of amphiphilic block copolymer for sustainable use.

Surface modification of electrically neutral hydrophilic polymers is one of the most promising methods for preventing biofouling and biological contamination by proteins and bacteria. Surface modification of inorganic materials such as silica-based glass can render them more durable and thus help in achieving the sustainable development goals. This study reports a novel method for the simple and effective surface modification of glass surfaces with amphiphilic block copolymers possessing the silane coupling segment composed of 3-(methacryloyloxy)propyltris (trimethylsilyloxy) silane and 3-methacryloxypropyltrimethoxysilane. The ability of hydrophilic segments composed of either 2-methacryloyloxyethyl phosphorylcholine (MPC) or poly(ethylene glycol) methyl ether methacrylate (mOEGMA) to prevent bacterial adhesion was investigated. The target block copolymers were prepared by reversible addition-fragmentation chain transfer polymerization and the monomer units of the hydrophilic segments were controlled to be either 120 or 160. The polymers were modified on the substrate by dip-coating. Contact angle measurements indicated that the block copolymer with the PMPC hydrophilic segment formed a hydrophilic surface without pre-hydration, while those with the PmOEGMA hydrophilic segment-coated surface became hydrophilic upon immersion in water. The block copolymer-coated surfaces decreased S. aureus adhesion, and a significant reduction was observed with the MPC-type block copolymer. The following surface design guidelines were thus concluded: (1) the block copolymer is superior to the random copolymer and (2) increasing the hydrophilic segment length further decreases bacterial adhesion.

Preparation of cellular membrane-mimicking glycopolymer interfaces by a solvent-assisted method on QCM-D sensor chips and their molecular recognition.

Carbohydrate-based membranes that show molecular recognition ability are interesting mimics of biointerfaces. Herein, we prepared glycopolymer membranes on QCM-D sensor chips using a solvent-assisted method and investigated their interactions with a target lectin. The membrane containing the glycopolymer with a random arrangement of the carbohydrate units adsorbed more lectin than that containing the glycopolymer with an organized block of carbohydrate units.

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

Bactericidal Ability of Well-Controlled Cationic Polymer Brush Surfaces and the Interaction Analysis by Quartz Crystal Microbalance with Dissipation.

In this study, cationic poly(2-(methacryloyloxy)ethyl) trimethylammonium chloride) (PMTAC) brush surfaces were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP), and their properties were systematically investigated to discuss the factors affecting their bactericidal properties and interactions with proteins. Model equations for the analysis of electrophoretic behaviors were considered for accurate parameter estimation to indicate the charge density at the interface. The zeta potential dependency of the PMTAC brushes was successfully analyzed using Smolchowski's equation and the Gouy-Chapman model, which describes the diffusive electric double layer. The analysis of the quartz crystal microbalance with dissipation (QCM-D) indicated that the electrostatic interaction promoted protein adsorption, with a large quantity of a negatively charged protein, bovine serum albumin (BSA), being adsorbed. The bactericidal efficiency of the high-graft-density polymer brush (0.45 chains nm-2) was higher than that of the low-graft-density polymer brush (0.06 chains nm-2). To investigate the mechanism of this phenomenon, we applied the dissipation change (ΔD) of QCM-D analysis. The BSA was likewise adsorbed when the brush structure was changed; however, the negative ΔD indicated that the BSA-adsorbed, high-graft-density PMTAC brush became a rigid state. In the bacteria culture media, the behaviors were the same as BSA adsorption, and the high-graft-density polymer brush was also estimated to be more rigid than the low-graft-density polymer brush. Moreover, for S. aureus adhesion after incubating in TSB, a small slope of ΔD/ΔF plots considered initial adsorption of bacteria on the high-graft-density polymer brush strongly interacted compared to that of the low-graft-density polymer brush. The scattered value of the slope of ΔD/ΔF on the high-graft-density polymer brush was considered to be due to the dead bacteria between the bacteria and the polymer brush interface. These investigations for a well-defined cationic polymer brush will contribute to the design of antibacterial surfaces.

An automatic immuno-microfluidic system integrating electrospun polystyrene microfibrous reactors for rapid detection of salivary cortisol.

Conventional competitive enzyme-linked immunosorbent assay (ELISA) to measure the cortisol level in body fluid consumes a large amount of time, owing to complicated operations involved and requirement for precise control of reagent addition. We developed an automatic microfluidic system to detect salivary cortisol rapidly, and an electrospun polystyrene (PS) microfiber-based reactor providing considerable binding sites for antibody immobilization, thus resolving the time limitations of competitive ELISA. Cortisol sample, horseradish peroxidase (HRP)-conjugated cortisol, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate were delivered to the PS reactor from containers in sequence by pumps automatically. The color variation due to oxidized TMB complex reflects the cortisol concentration level measured using an RGB phototransistor. In addition, the entire procedure from sample introduction to obtaining the photocurrent took only 15 min. This system can be implemented to quantify cortisol from 0.37 ng/mL to 30 ng/mL, and the limit of detection was estimated at 0.37 ng/mL.

Multi-Armed Star-Shaped Block Copolymers of Poly(ethylene glycol)-Poly(furfuryl glycidol) as Long Circulating Nanocarriers.

Multi-arm star-shaped block copolymers with precisely tuned nano-architectures are promising candidates for drug delivery. Herein, we developed 4- and 6-arm star-shaped block copolymers consisting of poly(furfuryl glycidol) (PFG) as the core-forming segments and biocompatible poly(ethylene glycol) (PEG) as the shell-forming blocks. The polymerization degree of each block was controlled by adjusting the feeding ratio of a furfuryl glycidyl ether and ethylene oxide. The size of the series of block copolymers was found to be less than 10 nm in DMF. In water, the polymers showed sizes larger than 20 nm, which can be related to the association of the polymers. The star-shaped block copolymers effectively loaded maleimide-bearing model drugs in their core-forming segment with the Diels-Alder reaction. These drugs were rapidly released upon heating via a retro Diels-Alder step. When the star-shaped block copolymers were injected intravenously in mice, they showed prolonged blood circulation, with more than 80% of the injected dose remaining in the bloodstream at 6 h after intravenous injection. These results indicate the potential of the star-shaped PFG-PEG block copolymers as long-circulating nanocarriers.

Cell Spheroid Formation on the Surface of Multi-Block Copolymers Composed of Poly(2-methoxyethyl acrylate) and Polyethylene Glycol.

3D structured cells have great drug screening potential because they mimic in vivo tissues better than 2D cultured cells. In this study, multi-block copolymers composed of poly(2-methoxyethyl acrylate) (PMEA) and polyethylene glycol (PEG) are developed as a new kind of biocompatible polymers. PEG imparts non-cell adhesion while PMEA acts as an anchoring segment to prepare the polymer coating surface. The multi-block copolymers show higher stability in water than PMEA. A specific micro-sized swelling structure composed of a PEG chain is observed in the multi-block copolymer film in water. A single NIH3T3-3-4 spheroid is formed in 3 h on the surface of the multi-block copolymers with 8.4 wt% PEG. However, at a PEG content of 0.7 wt%, spheroid formed after 4 days. The adenosine triphosphate (ATP) activity of cells and the internal necrotic state of the spheroid change depending on PEG loading in the multi-block copolymers. As the formation rate of cell spheroid on low-PEG-ratio multi-block copolymers is slow, internal necrosis of cell spheroid is less likely to occur. Consequently, the cell spheroid formation rate by changing the PEG chain content in multi-block copolymers is successfully controlled. These unique surfaces are suggested to be useful for 3D cell culture.

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip.

Here, we present a methodology based on multiplexed fluorescence screening of two- or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or immune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluorescence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.

Silver-loaded carboxymethyl cellulose nonwoven sheet with controlled counterions for infected wound healing.

Carboxymethyl cellulose (CMC) is a promising material for moist wound healing, and silver loading onto CMC has been examined for anti-bacterial activity. In this study, we developed silver-loaded CMC nonwoven sheets with different counterions, namely sodium CMC (CMC-Na/Ag) and partially protonated CMC (CMC-H/Ag), to examine their anti-bacterial and wound-healing properties. Owing to the presence of counter protons, CMC-H/Ag showed slower water adsorption, dissolution, and Ag release than CMC-Na/Ag. In addition, CMC-H/Ag and CMC-Na/Ag exhibited differences in anti-bacterial activities in shake-flask and inhibition zone tests in vitro. An in vivo experiment using a pressure ulcer mouse model with Pseudomonas aeruginosa infection showed that CMC-Na/Ag significantly accelerated wound healing compared to CMC-H/Ag and a commercially available Ag-loaded CMC nonwoven sheet, Aquacel Ag. These results suggest the importance of controlling CMC counterions and the therapeutic potential of the developed product as a wound dressing.

Design of biointerfaces composed of soft materials using controlled radical polymerizations.

Soft interface materials have an immense potential for the improvement of biointerfaces, which are the interface of biological and artificially designed materials. Controlling the chemical and physical structures of the interfaces at the nanometer level plays an important role in understanding the mechanism of the functioning and its applications. Controlled radical polymerization (CRP) techniques, including atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization, have been developed in the field of precision polymer chemistry. It allows the formation of well-defined surfaces such as densely packed polymer brushes and self-assembled nanostructures of block copolymers. More recently, a novel technique to prepare polymers containing biomolecules, called biohybrids, has also been developed, which is a consequence of the advancement of CRP so as to proceed in an aqueous media with oxygen. This review article summarizes recent advances in CRP for the design of biointerfaces.

Cell Adhesion and Migration on Thickness Gradient Bilayer Polymer Brush Surfaces: Effects of Properties of Polymeric Materials of the Underlayer.

In the field of tissue engineering and biomaterials, controlling the surface properties and mechanical properties of scaffold materials is crucial and has attracted much attention. Here, two types of bilayer polymer brushes composed of a hydrophilic underlying layer and a cationic surface layer [made of poly(2-aminoethyl methacrylate)] with a thickness gradient were prepared by surface-initiated atom-transfer radical polymerization. To investigate the influence of the stiffness as a mechanical property of the polymer brush on cell behavior, the underlayer was prepared from either 2-methacryloyloxyethyl phosphorylcholine or oligo(ethylene glycol) methyl ether methacrylate, with the bilayers designated as gradient poly(2-methacryloyloxyethyl phosphorylcholine)-block-poly(2-aminoethyl methacrylate) [grad-pMbA] and gradient poly(oligo[ethylene glycol] methyl ether methacrylate)-block-poly(2-aminoethyl methacrylate) [grad-pEGbA], respectively. Characterization of these surfaces was performed by spectroscopic ellipsometry, X-ray reflectivity, and determination of the zeta potential, static contact angle, and force curve. These diblock copolymer brushes with a thickness gradient helped to distinguish the effects of the mechanical and surface properties of the brushes on cell behavior. The attachment and motility of L929 fibroblasts and epithelial MCF 10A cells on the fabricated brushes were then assessed. L929 cells had a round shape on the thin surface layer of grad-pMbA and spread well on thicker areas. In contrast, MCF 10A cells spread well in areas of any thickness of either grad-pMbA or grad-pEGbA. Single MCF 10A cells migrated randomly on grad-pMbA, whereas grouped cells started to climb up along the thickness gradient of grad-pMbA. In contrast, both single and grouped MCF 10A cells migrated randomly on grad-pEGbA. These thickness gradient diblock copolymer brushes are simple, reproducible, and reasonable platforms that can facilitate practical applications of biomaterials, for example, in tissue engineering and biomaterials.

pH-Responsive Water-Soluble Polymer Carriers for Cell-Selective Metabolic Sialylation Labeling.

Cell-surface sialic acids can be metabolically labeled and subsequently modified using bioorthogonal chemistry. The method has great potential for targeted therapy and imaging; however, distinguishing the sialylation of specific cells remains a major challenge. Here, we described a cell-selective metabolic sialylation labeling strategy based on water-soluble polymer carriers presented with pH-responsive N-azidoacetylmannosamine (ManNAz) release. 2-Methacryloyloxyethyl phosphorylcholine contributed to increased water solubility and reduced nonspecific attachment to cells. Lactobionic acid residues, used for cell selectivity, recognized overexpressed receptors on target hepatoma cells and mediated cellular internalization. ManNAz caged by acidic pH-responsive carbonated ester linkage on the polymer was released inside target cells and expressed as azido sialic acid. Additionally, longer copolymer carriers enhanced the metabolic labeling efficiency of sialylation. This approach provides a platform for cell-selective labeling of sialylation and can be applied to high-resolution bioimaging and targeted therapy.

Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids.

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

Fluorescent polymeric nanoparticle for ratiometric temperature sensing allows real-time monitoring in influenza virus-infected cells.

Temperature is a key indicator of infection and disease, however, it is difficult to measure at a cellular level. Nanoparticles are applied to measure the cellular temperature, and enhancement of the stability and reliability of the signal and higher biocompatibility are demanded. We have developed fluorescent polymeric nanoparticles loaded with temperature-sensitive units (as rhodamine B) and internal reference units (as coumarin) for imaging and ratiometric sensing of the cellular temperature in the physiological range. The fluorescence signal of the nanoparticles was stable in the bio-environment and the ratiometric sensing strategy could overcome the concentration effect of nanoparticles. The nanoparticles were endocytosed by cells and partially presented in mitochondria. The fluorescence intensity ratio of rhodamine B and coumarin using nanoparticles showed good linear correlations in buffer solutions, cell suspensions, and imaging of living cells. Using the fluorescent polymeric nanoparticles, the change of temperature of cells during influenza virus infection could be individually monitored.

Evaluation of bacterial adhesion strength on phospholipid copolymer films with antibacterial ability using microfluidic shear devices.

Biomimetic phospholipid copolymer films are known to possess antifouling properties against protein adsorption and biofilm formation. However, the interactions between bacterial cells and material surfaces are not fully understood. This work investigated the bacterial adhesion strength of phospholipid copolymer films using a shear stress-tunable microfluidic device. The copolymer, comprising 2-methacryloyloxyethyl phosphorylcholine (MPC), 3-methacryloxypropyl trimethoxysilane (MPTMSi), and 3-(methacryloyloxy) propyl-tris(trimethylsilyloxy) silane (MPTSSi), formed crosslinked films on glass substrates; the thickness of the coating film was controlled by the polymer concentration during dip-coating. Polymer films with two typical thicknesses, 20 and 40 nm (denoted as C-20 and C-40, respectively), were prepared on the bottom wall of the microfluidic device. After seeding S. aureus in the microfluidic device, several shear stresses were applied to evaluate the adhesion strength of the polymer films. S. aureus was found to have weaker adhesion strength on the C-40 surface than on the C-20 surface; numerous bacterial cells detached from the C-40 surface on application of identical shear stress. To mimic the presence of plasma protein, fibrinogen (Fg) was introduced into the device before performing the bacterial adhesion assay. The results showed that the adsorption of Fg promoted S. aureus adhesion and strong interactions under shear stress. However, the adhesion strength of S. aureus did not affect the Fg adsorption for both the C-20 and C-40 surfaces. Using the shear stress-tunable microfluidic device, we found that the adhesion of S. aureus on the thicker and softer phospholipid copolymer was weak, and the cells easily detached under high shear stress.

Ultrasound-Based Scaffold-Free Core-Shell Multicellular Tumor Spheroid Formation.

In cancer research and drug screening, multicellular tumor spheroids (MCTSs) are a popular model to bridge the gap between in vitro and in vivo. However, the current techniques to culture mixed co-culture MCTSs do not mimic the structural architecture and cellular spatial distribution in solid tumors. In this study we present an acoustic trapping-based core-shell MCTSs culture method using sequential seeding of the core and shell cells into microwells coated with a protein repellent coating. Scaffold-free core-shell ovarian cancer OVCAR-8 cell line MCTSs were cultured, stained, cleared and confocally imaged on-chip. Image analysis techniques were used to quantify the shell thickness (23.2 ± 1.8 µm) and shell coverage percentage (91.2 ± 2.8%). We also show that the shell thickness was evenly distributed over the MCTS cores with the exception of being slightly thinner close to the microwell bottom. This scaffold-free core-shell MCTSs formation technique and the analysis tools presented herein could be used as an internal migration assay within the MCTS or to form core-shell MCTS co-cultures to study therapy response or the interaction between tumor and stromal cells.

Correction: Microfluidic flow control on charged phospholipid polymer interface.

Correction for 'Microfluidic flow control on charged phospholipid polymer interface' by Yan Xu et al., Lab Chip, 2007, 7, 199-206, DOI: 10.1039/B616851P.

"Nano": An Emerging Avenue in Electrochemical Detection of Neurotransmitters.

The growing importance of nanomaterials toward the detection of neurotransmitter molecules has been chronicled in this review. Neurotransmitters (NTs) are chemicals that serve as messengers in synaptic transmission and are key players in brain functions. Abnormal levels of NTs are associated with numerous psychotic and neurodegenerative diseases. Therefore, their sensitive and robust detection is of great significance in clinical diagnostics. For more than three decades, electrochemical sensors have made a mark toward clinical detection of NTs. The superiority of these electrochemical sensors lies in their ability to enable sensitive, simple, rapid, and selective determination of analyte molecules while remaining relatively inexpensive. Additionally, these sensors are capable of being integrated in robust, portable, and miniaturized devices to establish point-of-care diagnostic platforms. Nanomaterials have emerged as promising materials with significant implications for electrochemical sensing due to their inherent capability to achieve high surface coverage, superior sensitivity, and rapid response in addition to simple device architecture and miniaturization. Considering the enormous significance of the levels of NTs in biological systems and the advances in sensing ushered in with the integration of nanotechnology in electrochemistry, the analysis of NTs by employing nanomaterials as interface materials in various matrices has emerged as an active area of research. This review explores the advancements made in the field of electrochemical sensors for the sensitive and selective determination of NTs which have been described in the past two decades with a distinctive focus on extremely innovative attributes introduced by nanotechnology.