Poly(ethylene oxide) Concentration Gradient-Based Microfluidic Isolation of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have emerged as promising circulating biomarkers for non-invasive cancer diagnosis and management. Isolation and detection of CTCs in clinical samples are challenging due to the extreme rarity and high heterogeneity of CTCs. Here, we describe a poly(ethylene oxide) (PEO) concentration gradient-based microfluidic method for rapid, label-free, highly efficient isolation of CTCs directly from whole blood samples. Stable concentration gradients of PEO were formed within the microchannel by co-injecting the side fluid (blood sample spiked with 0.025% PEO) and center fluid (0.075% PEO solution). The competition between the elastic lift force and the inertial lift force enabled size-based separation of large CTCs and small blood cells based on their distinct migration patterns. The microfluidic device could process 1 mL of blood sample in 30 min, with a separation efficiency of >90% and an enrichment ratio of >700 for tumor cells. The isolated CTCs from blood samples were enumerated by immunofluorescence staining, allowing for discrimination of breast cancer patients from healthy donors with an accuracy of 84.2%. The concentration gradient-based microfluidic separation provides a powerful tool for label-free isolation of CTCs for a wide range of clinical applications.

Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage-Based Thermophoretic Assay.

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.

One-Step Thermophoretic AND Gate Operation on Extracellular Vesicles Improves Diagnosis of Prostate Cancer.

Circulating extracellular vesicles (EVs) have emerged as a valuable source of cancer biomarkers. However, the high degree of EV heterogeneity and the complexity of clinical samples pose a challenge in the sensitive identification of tumor-derived EVs. Here we introduce a one-step thermophoretic AND gate operation (Tango) assay that integrates polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs and simultaneous AND gate operation on EV membranes by dual-aptamers recognition. By using the Tango assay to detect tumor-derived EVs with co-presence of EpCAM and PSMA directly from serum in a homogeneous, separation-free format, we can discriminate prostate cancer (PCa) patients from benign prostatic hyperplasia (BPH) patients in the diagnostic gray zone with an accuracy of 91 % in 15 min. Our approach streamlines EV enrichment and AND gate operation on EVs in a single assay, providing a rapid, straightforward, and powerful method for precise and non-invasive diagnosis of cancer.

Rapid One-Step Detection of Viral Particles Using an Aptamer-Based Thermophoretic Assay.

Rapid and sensitive identification of viral pathogens such as SARS-CoV-2 is a critical step to control the pandemic disease. Viral antigen detection can compete with gold-standard PCR-based nucleic acid diagnostics in terms of better reflection of viral infectivity and reduced risk of contamination from enzymatic amplification. Here, we report the development of a one-step thermophoretic assay using an aptamer and polyethylene glycol (PEG) for direct quantitative detection of viral particles. The assay relies on aptamer binding to the spike protein of SARS-CoV-2 and simultaneous accumulation of aptamer-bound viral particles in laser-induced gradients of temperature and PEG concentration. Using a pseudotyped lentivirus model, a limit of detection of ∼170 particles µL-1 (26 fM of the spike protein) is achieved in 15 min without the need of any pretreatment. As a proof of concept, the one-step thermophoretic assay is used to detect synthetic samples by spiking viral particles into oropharyngeal swabs with an accuracy of 100%. The simplicity, speed, and cost-effectiveness of this thermophoretic assay may expand the diagnostic tools for viral pathogens.

Molecular Identification of Tumor-Derived Extracellular Vesicles Using Thermophoresis-Mediated DNA Computation.

Molecular profiling of tumor-derived extracellular vesicles (tEVs) holds great promise for non-invasive cancer diagnosis. However, sensitive and accurate identification of tEVs is challenged by the heterogeneity of EV phenotypes which reflect different cell origins. Here we present a DNA computation device mediated by thermophoresis for detection of tEVs. The strategy leverages the aptamer-based logic gate using multiple protein biomarkers on single EVs as the input and thermophoretic accumulation to amplify the output signals for highly sensitive and specific profiling of tEVs. Employing this platform, we demonstrate a high accuracy of 97% for discrimination of breast cancer (BC) patients and healthy donors in a clinical cohort (n = 30). Furthermore, molecular phenotyping assessed by tEVs is in concordance with the results from tissue biopsy in BC patients. The thermophoresis-mediated molecular computation on EVs thus provides new opportunities for accurate detection and classification of cancers.

A Tubular DNA Nanodevice as a siRNA/Chemo-Drug Co-delivery Vehicle for Combined Cancer Therapy.

Using the DNA origami technique, we constructed a DNA nanodevice functionalized with small interfering RNA (siRNA) within its inner cavity and the chemotherapeutic drug doxorubicin (DOX), intercalated in the DNA duplexes. The incorporation of disulfide bonds allows the triggered mechanical opening and release of siRNA in response to intracellular glutathione (GSH) in tumors to knockdown genes key to cancer progression. Combining RNA interference and chemotherapy, the nanodevice induced potent cytotoxicity and tumor growth inhibition, without observable systematic toxicity. Given its autonomous behavior, exceptional designability, potent antitumor activity and marked biocompatibility, this DNA nanodevice represents a promising strategy for precise drug design for cancer therapy.

Thermophoretic Detection of Exosomal microRNAs by Nanoflares.

Exosomal microRNAs (miRNAs) are reliable and noninvasive biomarkers for the early diagnosis of cancer. Yet, accurate and feasible detection of exosomal miRNAs is often hampered by the low abundance of miRNAs in exosomes and the requirement for RNA extraction in large sample volumes. Here we show a thermophoretic sensor implemented with nanoflares for in situ detection of exosomal miRNAs, without resorting to either RNA extraction or target amplification. Thermophoretic accumulation of nanoflare-treated exosomes leads to an amplified fluorescence signal upon the binding of exosomal miRNAs to nanoflares, allowing for direct and quantitative measurement of exosomal miRNAs down to 0.36 fM in 0.5 µL serum samples. One of the best markers, exosomal miR-375, showed an accuracy of 85% for detection of estrogen receptor-positive breast cancer at early stages (stages I, II). This work provides a feasible tool to improve the diagnosis of cancer.

Microfluidic Sonication To Assemble Exosome Membrane-Coated Nanoparticles for Immune Evasion-Mediated Targeting.

Using natural membranes to coat nanoparticles (NPs) provides an efficient means to reduce the immune clearance of NPs and improve their tumor-specific targeting. However, fabrication of these drug-loaded biomimetic NPs, such as exosome membrane (EM)- or cancer cell membrane (CCM)-coated poly(lactic-co-glycolic acid) (PLGA) NPs, remains a challenging task owing to the heterogeneous nature of biomembranes and labor-intensive procedures. Herein, we report a microfluidic sonication approach to produce EM-, CCM-, and lipid-coated PLGA NPs encapsulated with imaging agents in a one-step and straightforward manner. Tumor cell-derived EM-coated PLGA NPs consisting of both endosomal and plasma membrane proteins show superior homotypic targeting as compared to CCM-PLGA NPs of similar sizes and core-shell structures in both in vitro and in vivo models. The underlying mechanism is associated with a significantly reduced uptake of EM-PLGA NPs by macrophages and peripheral blood monocytes, revealing an immune evasion-mediated targeting of EM-PLGA NPs to homologous tumors. Overall, this work illustrates the promise of using microfluidic sonication approach to fabricate biomimetic NPs for better biocompatibility and targeting efficacy.

Enantiomorphic Microvortex-Enabled Supramolecular Sensing of Racemic Amino Acids by Using Achiral Building Blocks.

Chiral analysis of bioactive molecules is of increasing significance in chemical and life sciences. However, the quantitative detection of a racemic mixture of enantiomers is a challenging task, which relies on complicated and time-consuming multiple steps of chiral derivatization, chiral separation, and spectroscopic measurement. Herein, we show that, without the use of chiral molecules or pretreatment steps, the co-assembly of amino acids with achiral TPPS4 monomers controlled by enantiomorphic microvortices allows quantitative detection of racemic or enantiomeric amino acids, through analysis of the sign and magnitude of supramolecular chirality in different outlets of a microfluidic platform. A model demonstrates that chiral microvortices can induce an initial chiral bias by bending the sheet structure, resulting in supramolecular self-assembly of TPPS4 and amino acids of compatible chirality by the self-sorting. This sensing system may find versatile applications in chiral sensing.

λ-DNA- and Aptamer-Mediated Sorting and Analysis of Extracellular Vesicles.

Extracellular vesicles (EVs) are heavily implicated in diverse pathological processes. Due to their small size, distinct biogenesis, and heterogeneous marker expression, isolation and detection of single EV subpopulations are difficult. Here, we develop a λ-DNA- and aptamer-mediated approach allowing for simultaneous size-selective separation and surface protein analysis of individual EVs. Using a machine learning algorithm to EV signature based on their size and marker expression, we demonstrate that the isolated microvesicles are more efficient than exosomes and apoptotic bodies in discriminating breast cell lines and Stage II breast cancer patients with varied immunohistochemical expression of HER2. Our method provides an important tool to assess the EV heterogeneity at the single EV level with potential value in clinical diagnostics.

Stimulus-Responsive Plasmonic Chiral Signals of Gold Nanorods Organized on DNA Origami.

In response to environmental variations, living cells need to arrange the conformational changes of macromolecules to achieve the specific biofunctions. Inspired by natural molecular machines, artificial macromolecular assemblies with controllable nanostructures and environmentally responsive functions can be designed. By assembling macromolecular nanostructures with noble metal nanoparticles, environmental information could be significantly amplified and modulated. However, manufacturing dynamic plasmonic nanostructures that are efficiently responsive to different stimuli is still a challenging task. Here we demonstrate a stimulus-responsive plasmonic nanosystem based on DNA origami-organized gold nanorods (GNRs). L-shaped GNR dimers were assembled on rhombus-shaped DNA origami templates. The geometry and chiral signals of the GNR nanoarchitectures respond to multiple stimuli, including glutathione reduction, restriction enzyme action, pH change, or photoirradiation. While the glutathione reduction or restriction enzyme caused irreversible changes in the plasmonic circular dichroism (CD) signals, both pH and light irradiation triggered reversible changes in the plasmonic CD. Our system transduces external stimuli into conformational changes and circular dichroism responses in near-infrared (NIR) wavelengths. By this approach, programmable optical reporters for essential biological signals can be fabricated.

Double-Enzymes-Mediated Bioluminescent Sensor for Quantitative and Ultrasensitive Point-of-Care Testing.

We report an ultrasensitive, quantitative, and rapid bioluminescent immunosensor (ABS) for point-of-care testing (POCT) of the disease biomarker in clinical samples using double enzymes including alkaline phosphatase (ALP) and luciferase. In the presence of the biomarker, the ALP attached on the surface of immuno-nanocomplex dephosphorylates adenine triphosphate (ATP), subsequently inhibiting the ATP-luciferin-luciferase bioluminescent reaction. The highly sensitive response of ATP (picomolar level) allows for ultrasensitive detection of biomarker via the effective change of the bioluminescence intensity through ALP- and luciferase-catalyzed reactions, which can be quantitatively determined by a portable ATP detector. This ABS fulfills the criteria for POCT that performs sensitive (femtomolar level of biomarkers) and quantitative measurement quickly (less than 1 h) with minimal equipment (portable detector).

Integrated Microfluidic Platform with Multiple Functions To Probe Tumor-Endothelial Cell Interaction.

Interaction between tumor and endothelial cells could affect tumor growth and progression and induce drug resistance during cancer therapy. Investigation of tumor-endothelial cell interaction involves cell coculture, protein detection, and analysis of drug metabolites, which are complicated and time-consuming. In this work, we present an integrated microfluidic device with three individual components (cell coculture component, protein detection component, and pretreatment component for drug metabolites) to probe the interaction between tumor and endothelial cells. Cocultured cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) show higher resistance to chemotherapeutic agents than single-cultured cells, indicated by higher cell viability, increased expression of angiogenic proteins, and elevated level of paclitaxel metabolites under coculture conditions. This integrated microfluidic platform with multiple functions facilitates understanding of the interaction between tumor and endothelial cells, and it may become a promising tool for drug screening within an engineered tumor microenvironment.

One-Step Microfluidic Synthesis of Nanocomplex with Tunable Rigidity and Acid-Switchable Surface Charge for Overcoming Drug Resistance.

Multidrug resistance (MDR), is the key reason accounting for the failure of cancer chemotherapy, remains a dramatic challenge for cancer therapy. In this study, the one-step microfluidic fabrication of a rigid pH-sensitive micellar nanocomplex (RPN) with tunable rigidity and acid-switchable surface charge for overcoming MDR by enhancing cellular uptake and lysosome escape is demonstrated. The RPN is composed of a poly(lactic-co-glycolic acid) (PLGA) core and a pH-sensitive copolymer shell, which is of neutral surface charge during blood circulation. Upon internalization of RPN by cancer cells, the pH-responsive shell dissociates inside the acidic lysosomes, while the rigid and positively charged PLGA core improves the lysosomal escape. The cellular uptake and nuclear uptake of doxorubicin (Dox) from Dox-loaded RPN are 1.6 and 2.4 times higher than that from Dox-loaded pH-sensitive micelles (PM) using a Dox-resistant cancer model (MCF-7/ADR, re-designated NCI/ADR-RES) in vitro. Dox-loaded RPN significantly enhances the therapeutic efficacy (92% inhibition of tumor growth) against MCF-7/ADR xenograft tumor in mice, while Dox-loaded PM only inhibits the tumor growth by 36%. RPN avoids the use of complicated synthesis procedure of nanoparticle and the necessary to integrate multiple components, which can facilitate the clinical translation of this novel nanostructure.

Rotation-Facilitated Rapid Transport of Nanorods in Mucosal Tissues.

Mucus is a viscoelastic gel layer that typically protects exposed surfaces of the gastrointestinal (GI) tract, lung airways, and other mucosal tissues. Particles targeted to these tissues can be efficiently trapped and removed by mucus, thereby limiting the effectiveness of such drug delivery systems. In this study, we experimentally and theoretically demonstrated that cylindrical nanoparticles (NPs), such as mesoporous silica nanorods and calcium phosphate nanorods, have superior transport and trafficking capability in mucus compared with spheres of the same chemistry. The higher diffusivity of nanorods leads to deeper mucus penetration and a longer retention time in the GI tract than that of their spherical counterparts. Molecular simulations and stimulated emission of depletion (STED) microscopy revealed that this anomalous phenomenon can be attributed to the rotational dynamics of the NPs facilitated by the mucin fibers and the shear flow. These findings shed new light on the shape design of NP-based drug delivery systems targeted to mucosal and tumor sites that possess a fibrous structure/porous medium.

Sheathless Focusing and Separation of Diverse Nanoparticles in Viscoelastic Solutions with Minimized Shear Thinning.

Viscoelastic microfluidics becomes an efficient and label-free hydrodynamic technology to enrich and separate micrometer-scale particles, including blood cells, circulating tumor cells, and bacteria. However, the manipulation of nanoscale particles by viscoelastic microfluidics remains a major challenge, because the viscoelastic force acting on the smaller particle decreases dramatically. In contrast to the commonly used polymer solutions of high molecular weight, herein we utilize the aqueous solutions of poly(ethylene oxide) (PEO) of low molecular weight with minimized shear thinning but sufficient elastic force for high-quality focusing and separation of various nanoparticles. The focusing efficiencies of 100 nm polystyrene (PS) nanoparticles and λ-DNA molecules are 84% and 85%, respectively, in a double spiral microchannel, without the aid of sheath flows. Furthermore, we demonstrate the size-based viscoelastic separation of two sets of binary mixtures-100/2000 nm PS particles and λ-DNA molecules/blood platelets-all achieving separation efficiencies of >95% in the same device. Our proposal technique would be a promising approach for enrichment/separation of the nanoparticles encountered in applications of analytical chemistry and nanotechnology.

Nonspecific Organelle-Targeting Strategy with Core-Shell Nanoparticles of Varied Lipid Components/Ratios.

We report a nonspecific organelle-targeting strategy through one-step microfluidic fabrication and screening of a library of surface charge- and lipid components/ratios-varied lipid shell-polymer core nanoparticles. Different from the common strategy relying on the use of organelle-targeted moieties conjugated onto the surface of nanoparticles, here, we program the distribution of hybrid nanoparticles in lysosomes or mitochondria by tuning the lipid components/ratios in shell. Hybrid nanoparticles with 60% 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 20% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) can intracellularly target mitochondria in both in vitro and in vivo models. While replacing DOPE with the same amount of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the nanoparticles do not show mitochondrial targeting, indicating an incremental effect of cationic and fusogenic lipids on lysosomal escape which is further studied by molecular dynamics simulations. This work unveils the lipid-regulated subcellular distribution of hybrid nanoparticles in which target moieties and complex synthetic steps are avoided.

Nanocrystalline Cellulose-Assisted Generation of Silver Nanoparticles for Nonenzymatic Glucose Detection and Antibacterial Agent.

Nanocrystalline cellulose (NCC) is a kind of natural biopolymers with merits of large surface area, high specific strength and unique optical properties. This report shows that NCC can serve as the substrate, allowing glucose to reduce Tollen's reagent to produce silver nanoparticles (AgNPs) at room temperature. The generation of AgNPs is affected by the factors such as the concentrations of silver ions, NCC and glucose, as well as the different reaction temperatures. The AgNPs with NCC are applied for the development of a visual, quantitative, nonenzymatic and high-sensitive assay for glucose detection in serum. This assay is also used for monitoring the concentration change of glucose in medium during cell culture. For the antibacterial activity, the minimal inhibitory concentration (MIC) of the generated AgNPs with NCC is much lower than that of commercial AgNPs, attributed to the good dispersion of AgNPs with the presence of NCC. As NCC exhibits unique advantages including green, stable, inexpensive, and abundant, the NCC-based generation of AgNPs may find promising applications in clinical diagnosis, environmental monitoring, and the control of bacteria.

A microfluidic indirect competitive immunoassay for multiple and sensitive detection of testosterone in serum and urine.

We demonstrate a microfluidic-based indirect competitive chemiluminescence enzyme immunoassay (MIC) for multiple, sensitive, reliable and rapid detection of testosterone in human serum and urine samples. As MIC can detect biomarkers in a cost-effective and easy-to-operate manner, it may have great potential for clinical diagnosis and point-of-care testing (POCT).