Synthetic Cells from Droplet-Based Microfluidics for Biosensing and Biomedical Applications.

Synthetic cells function as biological mimics of natural cells by mimicking salient features of cells such as metabolism, response to stimuli, gene expression, direct metabolism, and high stability. Droplet-based microfluidic technology presents the opportunity for encapsulating biological functional components in uni-lamellar liposome or polymer droplets. Verified by its success in the fabrication of synthetic cells, microfluidic technology is widely replacing conventional labor-intensive, expensive, and sophisticated techniques justified by its ability to miniaturize and perform batch production operations. In this review, an overview of recent research on the preparation of synthetic cells through droplet-based microfluidics is provided. Different synthetic cells including lipid vesicles (liposome), polymer vesicles (polymersome), coacervate microdroplets, and colloidosomes, are systematically discussed. Efforts are then made to discuss the design of a variety of microfluidic chips for synthetic cell preparation since the combination of microfluidics with bottom-up synthetic biology allows for reproductive and tunable construction of batches of synthetic cell models from simple structures to higher hierarchical structures. The recent advances aimed at exploiting them in biosensors and other biomedical applications are then discussed. Finally, some perspectives on the challenges and future developments of synthetic cell research with microfluidics for biomimetic science and biomedical applications are provided.

Osmotic-Induced Reconfiguration and Activation in Membranized Coacervate-Based Protocells.

The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.

A Versatile Integrated Microfluidic Chip Based on Sonic Toothbrush-Assisted Mixing for Analyses of Diverse Biomolecules.

Usually, different assays and instrumentation are required for different types of targets, e.g., nucleic acids, proteins, small molecules, etc., because of significant differences in their structures and sizes. To increase efficiency and reduce costs, a desirable solution is to develop a versatile platform suitable for diverse objectives. Here, we established a versatile detection technique: first, target separation and enrichment were carried out using magnetic beads (MBs); then, different targets were converted to same barcoded DNA strands (BDs) released from gold nanoparticles; finally, sensitive detection of three different targets (miRNA-21, digoxigenin antibody, and aflatoxin B1) was achieved through exonuclease III (Exo III) cyclic cleavage-assisted signal amplification. To simplify the operation, we integrated this technique into a microfluidic chip with multiple chambers in which the requisite reagents were prestored. Just by moving the MBs through different chambers with a magnet, multiple steps can be completed. Due to the limited space in microfluidic chips, the full mixing of MBs and solution is a key point to improve reaction efficiency. The mixing can be achieved by acoustic vibration generated by a small, portable sonic toothbrush. Based on the microfluidic chip, the detection limits of the above three targets were 0.76 pM, 0.16 ng/mL, and 0.56 nM, respectively. Furthermore, miRNA-21 and Digoxigenin antibody (Dig-Ab) in serum and AFB1 in corn powder were also used to demonstrate the performance of this chip. Our versatile platform is easy to operate and is expected to develop into an automatic "sample-to-answer" device.

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication.

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H+ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.

Enhancing the Sensitivity of DNA and Aptamer Probes in the Dextran/PEG Aqueous Two-Phase System.

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

Invasion and Defense Interactions between Enzyme-Active Liquid Coacervate Protocells and Living Cells.

The design and construction of mutual interaction models between artificial microsystems and living cells have the potential to open a wide range of novel applications in biomedical and biomimetic technologies. In this study, an artificial form of invasion-defense mutual interactions is established in a community of glucose oxidase (GOx)-containing liquid coacervate microdroplets and living cells, which interact via enzyme-mediated reactive oxygen species (ROS) damage. The enzyme-containing coacervate microdroplets, formed via liquid-liquid phase separation, act as invader protocells to electrostatically bind with the host HepG2 cell, resulting in assimilation. Subsequently, the glucose oxidation in the liquid coacervates initiates the generation of H2 O2 , which serves as an ROS resource to block cell proliferation. As a defense strategy, introduction of catalase (CAT) into the host cells is exploited to resist the ROS damage. CAT-mediated decomposition of H2 O2 leads to the ROS scavenging and results in the recovery of cell viability. The results obtained in the current study highlight the remarkable opportunities for the development of mutual interacting communities on the interface of artificial protocells/living cells. They also provide a new approach for engineering cellular behaviors through exploiting artificial nonliving microsystems.

Liposome-Boosted Peroxidase-Mimicking Nanozymes Breaking the pH Limit.

Peroxidase-mimicking nanozymes such as Fe3 O4 nanoparticles are promising substitutes for natural enzymes like horseradish peroxidase. However, most such nanozymes work efficiently only in acidic conditions. In this work, the influence of various liposomes on nanozyme activity was studied. By introducing negatively charged liposomes, peroxidase-mimicking nanozymes achieved oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in neutral and even alkaline conditions, although the activity towards anionic 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was inhibited. The Fe3 O4 nanoparticles adsorbed on the liposomes without disrupting membrane integrity as confirmed by fluorescence quenching, dye leakage assays, and cryo-electron microscopy. Stabilization of the blue-colored oxidized products of TMB by electrostatic interactions was believed to be the reason for the enhanced activity. This work has introduced lipids to nanozyme research, and it also has practically important applications for using nanozymes at neutral pH, such as the detection of hydrogen peroxide and glucose.

Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability.

A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 µm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.

Use of ß-cyclodextrin-tethered cationic polymer based fluorescence enhancement of pyrene and hybridization chain reaction for the enzyme-free amplified detection of DNA.

Herein, we proposed an enzyme-free strategy for the amplified detection of DNA by combining the efficient fluorescence enhancement capability of a ß-cyclodextrin-tethered cationic polymer (cationic polyß-CD) to pyrene with the amplification capability of target DNA triggered hybridization chain reaction (HCR). Cationic polyß-CD with positive charge was synthesized. Two hairpin probes, H1 and H2, were employed in the system and the pyrene-labelled H2 was chosen as the signal unit. The pyrene attached on the sticky end of H2 was flexible and there was strong electrostatic interaction between cationic polyß-CD and negatively-charged H2, so pyrene could easily enter the cavity of CD that is tethered on the cationic polymer, accompanied by significant fluorescence enhancement. Once target DNA was introduced, HCR was triggered to form a rigid long dsDNA polymer with pyrene attached on it. The pyrene was hardly able to enter the cavity of cationic polyß-CD because of steric hindrance, leading to a weak fluorescent signal. Owing to the efficient pyrene fluorescence enhancement of cationic polyß-CD and the amplified capability of HCR, an enzyme-free sensitive detection of target DNA was achieved with a detection limit of 0.1 nM and high selectivity.

Competitive host-guest interaction between ß-cyclodextrin polymer and pyrene-labeled probes for fluorescence analyses.

We developed a novel homogeneous fluorescence analysis based on a novel competitive host-guest interaction (CHGI) mechanism between ß-cyclodextrin polymer (polyß CD) and pyrene-labeled probe for biochemical assay. Pyrene labeling with oligonucleotide strands can be recruited and reside in lipophilic cavities of polyß CD. This altered lipophilic microenvironment provides favored polarity for enhanced quantum efficiencies and extraordinarily increases the luminescence intensity of pyrene. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of polyß CD. The release of pyrene from polyß CD, which are followed by fluorescence extinguishing, will provide the clear signal turn-off in the presence of target DNA. We also introduced Exodeoxyribonuclease I (Exo I) and Exodeoxyribonuclease III (Exo III) to improve the sensitivity of this system, and the following product of cleavage reaction, pyrene-nucleotide, could more easily host-guest interact with polyß CD and emit stronger fluorescence than pyrene-labeled probe. In addition, the successful detection of adenosine is also demonstrated by using the similar sensing scheme. Although this scheme might be easily interfered by some biomolecules in the real test sample, it holds promising potential for detecting a broad range of other types of aptamer-binding chemicals and biomolecules.

A multiple amplification strategy for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer and pyrene.

A multiple amplification strategy has been developed for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer (ß-CDP) and pyrene. Briefly, the detection system consists of three parts: the polymerase and nicking enzyme-assisted isothermal strand displacement amplification (SDA) activated by a target DNA or microRNA; the exonuclease III-aided cyclic enzymatic amplification (CEA); and the fluorescence enhancement effect based on host-guest interaction between ß-CDP and pyrene. This strategy showed a good positive linear correlation with target DNA concentrations in the range from 75 fM to 1 pM with a detection limit of 41 fM. Significantly, our amplification platform was further validated and evaluated successfully by assaying miRNA-21 in human serum. The proposed assay has great potential as a nucleic acid quantification method for use in biomedical research, clinical analysis and disease diagnostics.

Visual and portable strategy for copper(II) detection based on a striplike poly(thymine)-caged and microwell-printed hydrogel.

Due to its importance to develop strategies for copper(II) (Cu(2+)) detection, we here report a visual and portable strategy for Cu(2+) detection based on designing and using a strip-like hydrogel. The hydrogel is functionalized through caging poly(thymine) as probes, which can effectively template the formation of fluorescent copper nanoparticles (CuNPs) in the presence of the reductant (ascorbate) and Cu(2+). On the hydrogel's surface, uniform wells of microliter volume (microwells) are printed for sample-injection. When the injected sample is stained by Cu(2+), fluorescent CuNPs will be in situ templated by poly T in the hydrogel. With ultraviolet (UV) irradiation, the red fluorescence of CuNPs can be observed by naked-eye and recorded by a common camera without complicated instruments. Thus, the strategy integrates sample-injection, reaction and indication with fast signal response, providing an add-and-read manner for visual and portable detection of Cu(2+), as well as a strip-like strategy. Detection ability with a detectable minimum concentration of 20 µM and practically applicable properties have been demonstrated, such as resistance to environmental interference and good constancy, indicating that the strategy holds great potential and significance for popular detection of Cu(2+), especially in remote regions. We believe that the strip-like hydrogel-based methodology is also applicable to other targets by virtue of altering probes.

Coacervate Microdroplets as Synthetic Protocells for Cell Mimicking and Signaling Communications.

Synthetic protocells are minimal systems that mimic certain properties of natural cells and are used to research the emergence of life from a nonliving chemical network. Currently, coacervate microdroplets, which are formed via liquid-liquid phase separation, are receiving wide attention in the context of cell biology and protocell research; these microdroplets are notable because they can provide liquid-like compartment structures for biochemical reactions by creating highly macromolecular crowded local environments. In this review, an overview of recent research on the formation of coacervate microdroplets through phase separation; the design of coacervate-based stimuli-responsive protocells, multichamber protocells, and membranized protocells; and their cell mimic behaviors, is provided. The simplified protocell models with precisely defined and tunable compositions advance the understanding of the requirements for cellular structure and function. Efforts are then discussed to establish signal communication systems in protocell and protocell consortia, as communication is a fundamental feature of life that coordinates matter exchanges and energy fluxes dynamically in space and time. Finally, some perspectives on the challenges and future developments of synthetic protocell research in biomimetic science and biomedical applications are provided.

Uncovering mechanisms governing stem growth in peanut (Arachis hypogaea L.) with varying plant heights through integrated transcriptome and metabolomics analyses.

The mechanisms responsible for stem growth in peanut (Arachis hypogaea L.) cultivars with varying plant heights remain unclear, despite the significant impact of plant height on peanut yield. Therefore, this study aimed to investigate the underlying mechanisms of peanut stem growth using phenotypic, physiological, transcriptomic, and metabolomic analyses. The findings revealed that the tallest cultivar, HY33, exhibited the highest rate of stem growth and accumulated the most stem dry matter, followed by the intermediate cultivar, SH108, while the dwarf cultivar, Df216, displayed the lowest values. Furthermore, SH108 exhibited a higher harvest index, as well as superior pod and kernel yields compared to both HY33 and Df216. Transcriptome and metabolome analyses identified differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) associated with phenylpropanoid and flavonoid biosynthesis. Notably, downregulated DEGs in Df216/HY33 and Df216/SH108 included phenylalanine ammonia-lyase (PAL), caffeoyl-CoA O-methyltransferase (COMT), and ferulate-5-hydroxylase (F5H), while downregulated DEMs included p-coumaryl alcohol, chlorogenic acid, and L-epicatechin. Compared to HY33, the reduced activities of PAL, COMT, and F5H resulted in a decreased stem lignin content in Df216. Additionally, downregulated DEGs involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis were identified in Df216/HY33, which contributed to the lowest levels of GA1, GA3, and BR contents in Df216. The results suggest that the dwarf phenotype arises from impaired GA and BR biosynthesis and signaling, resulting in a slower stem growth rate and reduced lignin accumulation.

Portable detection of multiple mycotoxins based on a sonic toothbrush, microfluidic chip and smartphone.

A portable method for on-site detection of three mycotoxins was developed based on a sonic toothbrush, microfluidic chip and smartphone. Our method could complete all procedures, including sample pretreatment, signal conversion and processing, without any sophisticated instruments. The limits of detection for these mycotoxins were lower than the limit values in cereals in the standards of China and the European Union.

Enzyme-active liquid coacervate microdroplets as artificial membraneless organelles for intracellular ROS scavenging.

Artificial organelles are microcompartments capable of performing catalytic reactions in living cells to replace absent or lost cellular functions. Coacervate microdroplets, formed via liquid-liquid phase separation, have been developed as membraneless organelles that mimic the dynamical organization of liquid organelles. However, the further studies focusing on cellular implanting of coacervate microdroplets in living cells to supplement the dysfunction of natural cells are still rare. Here catalase (CAT)-containing coacervate microdroplets, developed as artificial membraneless organelles with unique liquid compartments, were integrated into living cells to scavenge intracellular massive reactive oxygen species (ROS) and recover cell viability. The enzyme-containing coacervate microdroplets were constructed by sequestering CAT in poly(dimethyldiallylammonium chloride) (PDDA)/polyacrylic acid (PAA) coacervate microdroplets; their liquid-like fluidity was revealed by fluorescence recovery after bleaching, and coalescence experiment in vitro and in living cells. After cellular internalization, the coacervate microdroplets remained in the polymer-rich dense phase and retained enzymatic activities. CAT-mediated H2O2 removal and ROS scavenging in living cells decreased the cytotoxicity of H2O2, improving cell viability. The cell internalization of coacervate microdroplets in vitro provides a novel approach for designing artificial membraneless organelles in living cells. The strategy of using artificial organelle-mediated enzymatic reactions to supplement cellular dysfunctions can be exploited for their further biomedical applications.

DNA nanotubes in coacervate microdroplets as biomimetic cytoskeletons modulate the liquid fluidic properties of protocells.

Coacervate microdroplets, formed via liquid-liquid phase separation, have been proposed as a compartment model for the construction of artificial cells or organelles. However, these microsystems are very fragile and demonstrate liquid-like fluidity. Here, an artificial cytoskeleton based on DNA nanotubes was constructed in coacervate microdroplets to modulate the liquid fluidic properties of the microdroplets. The coacervate microdroplets were obtained from the association of oppositely charged polyelectrolytes through liquid-liquid phase separation, and DNA nanotubes were constructed by molecular tile self-assembly from six clip sequences. The DNA nanotubes were efficiently sequestered in the liquid coacervate microdroplets, and the rigid structure of the DNA nanotubes was capable of modulating the liquid fluidic properties of the coacervate protocell models, as indicated by coalescence imaging and atomic force microscopy analysis. Therefore, artificial cytoskeletons made from DNA nanotubes worked in modulating the liquid fluidic properties of coacervate microdroplets, in a manner akin to the cytoskeleton in the cell. DNA cytoskeletons have the potential to become an ideal platform with which how the liquid fluidic properties of cells are modulated by their cytoskeletons can be investigated, and the cell-sized coacervate microdroplets containing artificial cytoskeletons might be critical in developing a stable liquid-phase protocell model.

Signal processing and generation of bioactive nitric oxide in a model prototissue.

The design and construction of synthetic prototissues from integrated assemblies of artificial protocells is an important challenge for synthetic biology and bioengineering. Here we spatially segregate chemically communicating populations of enzyme-decorated phospholipid-enveloped polymer/DNA coacervate protocells in hydrogel modules to construct a tubular prototissue-like vessel capable of modulating the output of bioactive nitric oxide (NO). By decorating the protocells with glucose oxidase, horseradish peroxidase or catalase and arranging different modules concentrically, a glucose/hydroxyurea dual input leads to logic-gate signal processing under reaction-diffusion conditions, which results in a distinct NO output in the internal lumen of the model prototissue. The NO output is exploited to inhibit platelet activation and blood clot formation in samples of plasma and whole blood located in the internal channel of the device, thereby demonstrating proof-of-concept use of the prototissue-like vessel for anticoagulation applications. Our results highlight opportunities for the development of spatially organized synthetic prototissue modules from assemblages of artificial protocells and provide a step towards the organization of biochemical processes in integrated micro-compartmentalized media, micro-reactor technology and soft functional materials.

Formation of PdPt alloy nanodots on gold nanorods: tuning oxidase-like activities via composition.

The island growth mode of Pt was employed to guide the forma-tion of PdPt alloy nanodots on gold nanorods (Au@PdPt NRs). Well-defined alloy nanodots, with tunable Pd/Pt ratios from 0.2 to 5, distribute homogeneously on the surface of the Au NR. Formation of nanodots shell leads to the red-shift and broadening of the longitudinal surface plasmon resonance (LSPR) band of the Au NRs. The Au@PdPt alloy NRs exhibit catalytic activity toward oxidation of often-used chromogenic substrates by dissolved oxygen under mild conditions, suggesting a new type of oxidase mimics. Composition dependence catalytic activity is observed for the oxidation of ascorbic acid (AA) and 3,3',5,5'-tetramethylbenzidine (TMB) and for the reduction of p-nitrophenol. For AA and TMB, catalytic activity enhances quickly at lower Pd/Pt ratios and tends to saturate at higher Pd/Pt ratios. For p-nitrophenol reduction, catalytic activity shows a nice linear relationship with Pd/Pt ratio owing to much higher catalytic activity of Pd. In conclusion, proper alloying of Pd and Pt presents an effective route to tailor the catalytic activity. Interesting, alloy nanodots can also catalyze the oxidation of Fe (II) to Fe (III) by dissolved oxygen. Thus, based on the competitive oxidation of TMB and Fe (II), selective detection of the latter can be achieved.

Coacervate microdroplet protocell-mediated gene transfection for nitric oxide production and induction of cell apoptosis.

Liquid coacervate microdroplets have been widely explored as membrane-free compartment protocells for cargo delivery in therapeutic applications. In this study, coacervate protocells were developed as gene carriers for transfection of nitric oxide synthase (NOS) and overproduction of nitric oxide (NO) for killing of cancer cells. The coacervate microdroplet protocells were formed via the liquid-liquid phase separation of oppositely charged diethylaminoethyl-dextran/polyacrylic acids. The coacervate microdroplet protocells were found to facilitate gene transfection, which was demonstrated by cell imaging of the internalized coacervate microdroplets containing plasmids of enhanced green fluorescent protein. Due to their high transfection capability, the coacervate protocells were subsequently utilized for the delivery of NOS plasmids (pNOS). The cellular internalization of pNOS-containing coacervate carriers was found to result in high NOS expression coupled with NO overproduction, which then induced cell apoptosis and decreased cell viability. The cell apoptosis is associated with NO-mediated mitochondrial damage. The enhanced gene transfection was attributed to coacervate microdroplets' unique high sequestration capability and liquid-like fluidity. Overall, the incorporation of genes in coacervate microdroplets was demonstrated as a viable and novel strategy for the development of cargo biocarriers for biomedical applications.