Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy.

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

Atomic Force Microscopy Lifetime Analysis: An Intuitive Method for Evaluating Receptor Tyrosine Kinase Dimer-Targeting Inhibitors.

Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal-epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ1 = 207.87 ± 4.69 ms) and liganded MET dimers (τ2 = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ1 and τ2, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ2, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.

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.

Photocaged amplified FRET nanoflares: spatiotemporal controllable of mRNA-powered nanomachines for precise and sensitive microRNA imaging in live cells.

There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a 'one-to-more' ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.

DNA-Encoded Bidirectional Regulation of the Peroxidase Activity of Pt Nanozymes for Bioanalysis.

Rational regulation of nanozyme activity can promote biochemical sensing by expanding sensing strategies and improving sensing performance, but the design of effective regulatory strategies remains a challenge. Herein, a rapid DNA-encoded strategy was developed for the efficient regulation of Pt nanozyme activity. Interestingly, we found that the catalytic activity of Pt nanozymes was sequence-dependent, and its peroxidase activity was significantly enhanced only in the presence of T-rich sequences. Thus, different DNA sequences realized bidirectional regulation of Pt nanozyme peroxidase activity. Furthermore, the DNA-encoded strategy can effectively enhance the stability of Pt nanozymes at high temperatures, freezing, and long-term storage. Meanwhile, a series of studies demonstrated that the presence of DNA influenced the reduction degree of H2PtCl6 precursors, which in turn affected the peroxidase activity of Pt nanozymes. As a proof of application, the sensor array based on the Pt nanozyme system showed superior performance in the accurate discrimination of antioxidants. This study obtained the regulation rules of DNA on Pt nanozymes, which provided theoretical guidance for the development of new sensing platforms and new ideas for the regulation of other nanozyme activities.

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.

"One suction and one extrusion" mode-based wash-free platform for determination of breast cancer cell-derived exosomes.

A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particles⋅mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.

Size-Controllable and Self-Assembled DNA Nanosphere for Amplified MicroRNA Imaging through ATP-Fueled Cyclic Dissociation.

Visualizing intracellular microRNA (miRNA) is of great importance for revealing its roles in the development of disease. However, cell membrane barrier, complex intracellular environment and low abundance of target miRNA are three main challenges for efficient imaging of intracellular miRNA. Here, we report a size-controllable and self-assembled DNA nanosphere with ATP-fueled dissociation property for amplified miRNA imaging in live cells and mice. The DNA nanosphere was self-assembled from Y-shaped DNA (Y-DNA) monomers through predesigned base pair hybridization, and the size could be easily controlled by varying the concentration of Y-DNA. Once the nanosphere was internalized into cells, the intracellular specific target miRNA would trigger the cyclic dissociation of the DNA nanosphere driven by ATP, resulting in amplified FRET signal. The programmable DNA nanosphere has been proven to work well for detecting the expression of miRNA in cancer cells and in mice, which demonstrates its fairish cell penetration, stability and sensitivity.

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.

Giant Coacervate Vesicles As an Integrated Approach to Cytomimetic Modeling.

Although giant unilamellar vesicles (GUVs) have been extensively studied as synthetic cell-like microcompartments, their applicability as cytomimetic models is severely compromised by low levels of membrane permeability, low encapsulation efficiencies, and high physicochemical instability. Here, we develop an integrated cytomimetic model comprising a macromolecularly crowded interior with high sequestration efficiency and enclosed within a phospholipid membrane that is permeable to molecules below a molecular weight cutoff of ca. 4 kDa. The protocells are readily prepared by spontaneous assembly of a phospholipid membrane on the surface of preformed polynucleotide/polysaccharide coacervate microdroplets and are designated as giant coacervate vesicles (GCVs). Partial anchoring of the GCV membrane to the underlying coacervate phase results in increased robustness, lower membrane fluidity, and increased permeability compared with GUV counterparts. As a consequence, enzyme and ribozyme catalysis can be triggered in the molecularly crowded interior of the GCV but not inside the GUVs when small molecule substrates or inducers are present in the external environment. By integrating processes of membrane-mediated compartmentalization and liquid-liquid microphase separation, GCVs could offer substantial advantages as cytomimetic models, synthetic protocells, and artificial biomolecular microreactors.

Orderly Assembled, Self-Powered FRET Flares for MicroRNA Imaging in Live Cells.

Signal amplification provides unparalleled opportunities for visualizing low-abundance targets in living cells. However, self-powered signal amplification has not been achieved because of the lack of "fuel" in living cells. Thus, the aim of this work was to develop an integrated amplification platform for the detection of intracellular miRNA by itself. This system, termed self-powered FRET flares (SPF), was first established by self-assembly to form a DNA nanostructure, and then the FRET flares and fuel DNA as the driving force were precisely and orderly loaded on it, which was able to power target recycle and realize signal amplification without any auxiliary additives under the trigger of intracellular miRNA-21. In addition, it employed AS1411 aptamer to target specific cancer cells and facilitated cell internalization of assembly DNA nanostructures. As a proof of concept, we demonstrated that SPF enabled rapid response to miRNA-21 and improvement of the detection sensitivity compared to previously proposed FRET flares without amplification. This strategy is promising for advancing integrated and self-powered nanomachines to execute diverse tasks, facilitating their biological and medical application.

Ratiometric Fluorescent DNA Nanostructure for Mitochondrial ATP Imaging in Living Cells Based on Hybridization Chain Reaction.

For intracellular molecular detection, the appropriate probes should include the abilities to enter target cells noninvasively, target specific sites, and then respond to the analytes reliably. Herein, a ratiometric fluorescent DNA nanostructure (RFDN) was designed for mitochondrial adenosine triphosphate (ATP) imaging in living cells. The DNA nanostructure was constructed by continuous hybridization of two hairpin DNA strands (HS1-Cy3 and HS2-Cy5) under the initiation of the trigger. HS1-Cy3 and HS2-Cy5 contained split aptamer fragments of ATP and are labeled with a fluorescent donor (Cy3) and acceptor (Cy5), respectively. The RFDN integrated multiple split aptamer fragments and increased the local concentration of sensing probes. The binding of ATP to aptamer fragments on the RFDN shortened the distance between Cy3 and Cy5, resulting in obvious ratiometric signals (fluorescence resonance energy transfer). The RFDN showed good biocompatibility and can be internalized into cells in a caveolin-dependent endocytosis pathway. The co-localization imaging results indicated that the DNA nanostructure could target the mitochondria via Cy3 and Cy5. Moreover, the confocal imaging results showed that the intracellular ATP changes stimulated by drugs in living cells could be indicated by the RFDN. In this way, the RFDN is expected to be a simple, flexible, and general platform for chemo/biosensing in living cells.

DNA Hydrogelation-Enhanced Imaging Ellipsometry for Sensing Exosomal microRNAs with a Tunable Detection Range.

Conventional imaging ellipsometry-based biosensing faces the challenges of poor sensitivity and narrow dynamic range, especially for some small molecules such as microRNA. Given that detection of various exosomal miRNAs with tunable range could provide high-precision disease information and improve the accuracy of diagnosis, a sensitive imaging ellipsometry sensor was introduced to improve sensitivity with a tunable detection range by terminus-regulated DNA hydrogelation. Tetrahedron DNA probes with complementary sequence to the target miRNA were used as biorecognition elements to form DNA hydrogelation. This DNA hydrogelation was formed by template-independent and isothermal amplification on the Au film. Due to its high dielectric constant, DNA hydrogelation structure could be used for improving the sensitivity of imaging ellipsometry significantly. Importantly, by changing the cycle of the DNA hydrogelation amplification, this strategy showed a tunable detection range from fM to nM for miRNA with a limit of detection of 0.2 fM for let-7a, 10 fM for miR-375, and 40 pM for miR-21. Furthermore, it also performed satisfactorily for the miRNA sensing in 50% human serum and 50% human plasma. This DNA hydrogelation-enhanced imaging ellipsometry could broaden the applications of conventional imaging ellipsometry in biosensing and provide a sensitive method for sensing miRNAs at different abundances.

Lipophilic G-Quadruplex Isomers as Biomimetic Ion Channels for Conformation-Dependent Selective Transmembrane Transport.

Transport selectivity is a challenge in the design of biomimetic transmembrane channels. In the present study, specific ion-dependent lipophilic G-quadruplexes displaying different conformations were designed for the construction of highly selective artificial transmembrane channels. The presence of Pb2+ and K+ ions prompted the folding of lipophilic PS2.M sequence into G-quadruplexes with antiparallel and parallel conformations. Membrane immobilization of the G-quadruplex channels restricted the reversible configurational changes between different topologies, which was confirmed by Förster resonance energy transfer (FRET) analysis. 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport assays revealed that Pb2+-stabilized antiparallel isomers, and K+-stabilized parallel isomers exhibited significant differences in the transmembrane transport. The former showed high Pb2+ transport activity (EC50 = 1.55 µM) and selectivity (Pb2+/K+ selectivity = 30.6), while the latter demonstrated high K+ transport activity (EC50 = 0.56 µM) and selectivity (K+/Pb2+ selectivity = 31.8). The cation selectivity of these channel mimics was also consistent with the outcomes of the conducted fluorescent probe assays. The results described herein provide a platform for effective development of conformation-dependent ion-selective biomimetic transmembrane channels. The G-quadruplex channels demonstrate high potential for application in the fields of molecular diagnostics, logic biocomputing, selective separation, and single-molecule biosensing.

Engineering and Application of a Myoglobin Binding Split Aptamer.

Given that a split aptamer provides a chance for the development of a sandwich assay for targets with only one aptamer, it has received extensive attention in biosensing. However, due to the lack of binding mechanisms and reliable methods, there were still a few split aptamers that bind to proteins. In this work, cardiac biomarker myoglobin (Myo) was selected as a model, a new strategy of engineering split aptamers was explored with atomic force spectroscopy (AFM), and split aptamers against target protein could be achieved by choosing the optimal binding probability between split aptamers and target. Then, the obtained split aptamers were designed for Myo detection based on dynamic light scattering (DLS). The results demonstrated that the obtained split aptamers could be used to detect targets in human serum. The strategy of engineering split aptamers has the advantages of being intuitive and reliable and could be a general strategy for obtaining split aptamers.

Exploring Interactions of Aptamers with Aß40 Amyloid Aggregates and Its Application: Detection of Amyloid Aggregates.

The exhaustive investigating interactions between recognition probes and amyloid aggregates, especially simultaneous recognition events, are challenging and crucial for the design of biosensing probes and further diagnosis of amyloid diseases. In the present work, the interactions of aptamers (Apts) with ß-amyloid (Aß) aggregates were explored thoroughly by single-molecule force spectroscopy (SMFS). Indeed, it was found that the interaction of aptamer1 (Apt1)-amyloid aggregates was different from that of aptamer2 (Apt2)-Aß40 aggregates at the single-molecule level. Especially, the interaction force of Apt1-Aß40 fibril showed a double distinguishing Gaussian fitting. The only unimodal distribution of the force histogram was displayed for the interactions of Apt2-Aß40 oligomer, Apt2-Aß40 fibril, and Apt1-Aß40 oligomer. More intriguingly, two Apts could bind to amyloid aggregates simultaneously. With the assistance of two Apts recognition, a novel sensitive dual Apt-based surface plasmon resonance (SPR) sensor using Au nanoparticles (AuNPs) was developed for quantifying Aß40 aggregates. The dual Apt-based SPR sensor not only avoided the limitation of steric hindrance and epitope but also employed simple operation as well as inexpensive recognition probes. A detection limit as low as 0.2 pM for Aß40 oligomer and 0.05 pM for Aß40 fibril could be achieved. Moreover, the established sensor could be successfully applied to detect Aß40 aggregates in artificial cerebrospinal fluid (CSF) and undiluted real CSF. This work could provide a strategy to monitor a simultaneous recognition event using SMFS and broaden the application of Apts in the diagnosis of neurodegenerative diseases.

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.

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy.

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165 . On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165 . Besides, the dissociation rate constant (koff ) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165 , and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165 . It indicated that split aptamer also possessed high affinity with VEGF165 . The work can provide a new method for exploring the interaction of split aptamer/its targets at single-molecule level.

Engineering DNAzyme cascade for signal transduction and amplification.

Inspired by the natural enzyme cascade reaction, a multiple DNAzyme cascade platform is engineered to imitate the intracellular process of signal transduction and signal amplification. In this design, when particular stimuli appear, an activated upstream DNAzyme will cleave a well-designed intermediary S1, releasing a downstream DNAzyme that can cleave the reporter substrate S2 to output signals. Thus, the signal is passed from the upstream DNAzyme to the downstream DNAzyme through a well-designed intermediary, accomplishing signal transduction and signal amplification. According to the experimental results, the DNAzyme cascades are capable of improving sensitivity for bioassays compared with that for single DNAzyme-based biocatalysis, which holds promise for potential applications, such as biomolecular computing, logic circuits and precision medicine.

Photothermal and fluorescent dual-mode assay based on the formation of polydopamine nanoparticles for accurate determination of organophosphate pesticides.

A photothermal and fluorescent dual-mode assay for sensitive organophosphate pesticides (Ops) determination is reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. In the presence of ALP, ascorbic acid 2-phosphate (AAP) can be catalyzed to produce ascorbic acid (AA). AA can reduce MnO2 nanosheets, further inhibiting the oxidation of dopamine (DA). Ops as an inhibitor for ALP activity prevents the formation of AA and the reduction of MnO2 nanosheets. Eventually, the formation of PDA nanoparticles is promoted. The inhibitory effect of Ops on ALP activity causes obvious changes of photothermal signals and fluorescence signal at 495 nm. The detection limit (LOD) of dimethoate is 0.1 µM. The method displays excellent sensing capability for the dimethoate assay in real water with good recoveries of 99.4-107.6%. Graphical abstract A photothermal and fluorescent dual-mode biosensor for sensitive Ops detection was reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. The dual-mode method significantly improved the accuracy and reliability of the results.