Dual-Encoded Affinity Microbead Signature Combinatorial Profiling for Acute Myocardial Infarction High-Sensitivity Diagnosis.

The early diagnosis of acute myocardial infarction (AMI) is dependent on the combined feedback of multiple cardiac biomarkers. However, it remains challenging to precisely detect multicardiac biomarkers in complex blood early due to the lack of sensitive and specific diagnostic indicators and the low abundance and small size of associated biomarkers with high specificity (such as microRNAs). To make matters worse, spectral overlap significantly limits the multiplex analysis of cardiac biomarkers by fluorescent probes, leading to bias in the diagnosis of myocardial infarction. Herein, we developed a method for simultaneous detection of miRNAs and protein biomarkers using size- and color-coded microbeads that carry signature for target capture. We also constructed a microfluidic chip with different spacer arrays that segregate these microbeads in different chip regions according to their size to produce signature signals, indicating the level of different biomarkers. The signals on the microbeads were hugely amplified by catalytic hairpin assembly and rolling circle amplification. Notably, this strategy enables the simultaneous and in situ sensitive profiling of six kinds of biomarkers via adding two different fluorescent labels, removing the limitations of spectral overlap. We envision that the strategy has great potential for application in clinical diagnosis for AMI.

Micro-Engineered Organoid-on-a-Chip Based on Mesenchymal Stromal Cells to Predict Immunotherapy Responses of HCC Patients.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. Patient-derived organoid (PDO) has great potential in precision oncology, but low success rate, time-consuming culture, and lack of tumor microenvironment (TME) limit its application. Mesenchymal stromal cells (MSC) accumulate in primary site to support tumor growth and recruit immune cells to form TME. Here, MSC and peripheral blood mononuclear cells (PBMC) coculture is used to construct HCC organoid-on-a-chip mimicking original TME and provide a high-throughput drug-screening platform to predict outcomes of anti-HCC immunotherapies. HCC-PDOs and PBMC are co-cultured with MSC and Cancer-associated fibroblasts (CAF). MSC increases success rate of biopsy-derived PDO culture, accelerates PDO growth, and promotes monocyte survival and differentiation into tumor-associated macrophages. A multi-layer microfluidic chip is designed to achieve high-throughput co-culture for drug screening. Compared to conventional PDOs, MSC-PDO-PBMC and CAF-PDO-PBMC models show comparable responses to chemotherapeutic or targeted anti-tumor drugs but more precise prediction potential in assessing patients' responses to anti-PD-L1 drugs. Moreover, this microfluidic platform shortens PDO growth time and improves dimensional uniformity of organoids. In conclusion, the study successfully constructs microengineered organoid-on-a-chip to mimic TME for high-throughput drug screening, providing novel platform to predict immunotherapy response of HCC patients.

Application of microfluidic technologies on COVID-19 diagnosis and drug discovery.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has boosted the development of antiviral research. Microfluidic technologies offer powerful platforms for diagnosis and drug discovery for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and drug discovery. In this review, we introduce the structure of SARS-CoV-2 and the basic knowledge of microfluidic design. We discuss the application of microfluidic devices in SARS-CoV-2 diagnosis based on detecting viral nucleic acid, antibodies, and antigens. We highlight the contribution of lab-on-a-chip to manufacturing point-of-care equipment of accurate, sensitive, low-cost, and user-friendly virus-detection devices. We then investigate the efforts in organ-on-a-chip and lipid nanoparticles (LNPs) synthesizing chips in antiviral drug screening and mRNA vaccine preparation. Microfluidic technologies contribute to the ongoing SARS-CoV-2 research efforts and provide tools for future viral outbreaks.

Modular DNA-Origami-Based Nanoarrays Enhance Cell Binding Affinity through the "Lock-and-Key" Interaction.

Surface proteins of cells are generally recognized through receptor-ligand interactions (RLIs) in disease diagnosis, but their nonuniform spatial distribution and higher-order structure lead to low binding affinity. Constructing nanotopologies that match the spatial distribution of membrane proteins to improve the binding affinity remains a challenge. Inspired by the multiantigen recognition of immune synapses, we developed modular DNA-origami-based nanoarrays with multivalent aptamers. By adjusting the valency and interspacing of the aptamers, we constructed specific nanotopology to match the spatial distribution of target protein clusters and avoid potential steric hindrance. We found that the nanoarrays significantly enhanced the binding affinity of target cells and synergistically recognized low-affinity antigen-specific cells. In addition, DNA nanoarrays used for the clinical detection of circulating tumor cells successfully verified their precise recognition ability and high-affinity RLIs. Such nanoarrays will further promote the potential application of DNA materials in clinical detection and even cell membrane engineering.

Identifying the Phenotypes of Tumor-Derived Extracellular Vesicles Using Size-Coded Affinity Microbeads.

Tumor-derived extracellular vesicle (tEV) biomarkers can reflect cancer cell phenotypes and have great potential for cancer diagnosis and treatment. However, tEVs display high heterogeneity, and rapid and sensitive identification of EV biomarkers remains challenging due to their low expression. Spectral overlap also significantly limits the multiplex analysis of EV biomarkers by fluorescent probes. Herein, we developed a method for highly sensitive tEV phenotyping that uses size-coded microbeads that carry hairpin probes that can bind to aptamers targeting distinct tEV biomarkers. We also designed a microfluidic chip containing spacer arrays that segregate these microbeads in distinct chip regions according to their size to generate location-specific signals indicating the level of different EV biomarkers. The EV biomarker signal on these microbeads was amplified by in situ rolling cyclic amplification (RCA). This strategy permits the simultaneous detection of multiple tEV phenotypes by fluorescence spectroscopy without the limitations of spectral overlap. This study demonstrates that this tEV phenotyping method can rapidly and simultaneously detect six different tEV phenotypes with high sensitivity. Due to the programmability of the sensing platform, this method can be rapidly adapted to detect different tEV phenotype substitutions of the detected biomarkers. Notably, clinical cohort studies show that this strategy may provide new ideas for the precise diagnosis and personalized treatment of cancer patients.

In situ signal amplification improves the capture efficiency of circulating tumor cells with low expression of EpCAM.

Circulating tumor cells (CTCs) as non-invasive biomarkers have great potential in evaluating tumor progression and prognosis. However, effective enrichment of CTCs and minimizing phenotypic bias remain a serious challenge. Herein, a DNA tetrahedron-aptamer complex-mediated rolling circle amplification (TDN-RCA) strategy is developed for cell surface protein signal amplification and CTC enrichment, employing DNA tetrahedron-EpCAM aptamer complex as a scaffold and initiating rolling circle amplification (RCA) reaction on the surface of CTCs in situ. The DNA tetrahedron-aptamer complex enables the cell-specific recognition and enhances cell membrane anchoring ability, generating a large number of magnetic beads binding sites through the RCA reaction in situ. Thus, the signals of cell surface markers with low expression levels are amplified in situ and then efficient CTC enrichment is achieved. This method improves the capture efficiency of CTCs with low expression of EpCAM, which has great potential in clinical application.

A Dual-Encoded Bead-Based Immunoassay with Tunable Detection Range for COVID-19 Serum Evaluation.

Serological assay for coronavirus 2019 (COVID-19) patients including asymptomatic cases can inform on disease progression and prognosis. A detection method taking into account multiplex, high sensitivity, and a wider detection range will help to identify and treat COVID-19. Here we integrated color-size dual-encoded beads and rolling circle amplification (RCA) into a bead-based fluorescence immunoassay implemented in a size sorting chip to achieve high-throughput and sensitive detection. We used the assay for quantifying COVID-19 antibodies against spike S1, nucleocapsid, the receptor binding domain antigens. It also detected inflammatory biomarkers including interleukin-6, interleukin-1ß, procalcitonin, C-reactive protein whose concentrations range from pg mL-1 to µg mL-1 . Use of different size beads integrating with RCA results in a tunable detection range. The assay can be readily modified to simultaneously measure more COVID-19 serological molecules differing by orders of magnitude.

Pharmaceutical applications of framework nucleic acids.

DNA is a biological polymer that encodes and stores genetic information in all living organism. Particularly, the precise nucleobase pairing inside DNA is exploited for the self-assembling of nanostructures with defined size, shape and functionality. These DNA nanostructures are known as framework nucleic acids (FNAs) for their skeleton-like features. Recently, FNAs have been explored in various fields ranging from physics, chemistry to biology. In this review, we mainly focus on the recent progress of FNAs in a pharmaceutical perspective. We summarize the advantages and applications of FNAs for drug discovery, drug delivery and drug analysis. We further discuss the drawbacks of FNAs and provide an outlook on the pharmaceutical research direction of FNAs in the future.

Accurate Isolation of Circulating Tumor Cells via a Heterovalent DNA Framework Recognition Element-Functionalized Microfluidic Chip.

Detection of circulating tumor cells (CTCs) has provided a noninvasive and efficient approach for early diagnosis, treatment, and prognosis of cancer. However, efficient capture of CTCs in the clinical environment is very challenging because of the extremely rare and heterogeneous expression of CTCs. Herein, we fabricated a multimarker microfluidic chip for the enrichment of heterogeneous CTCs from peripheral blood samples of breast cancer patients. The multimarker aptamer cocktail DNA nanostructures (TP-multimarker) were modified on a deterministic lateral displacement (DLD)-patterned microfluidic chip to enhance the capture efficiency through the size selection effect of DLD arrays and the synergistic effect of multivalent aptamers. As compared to a monovalent aptamer-modified chip, the multimarker chip exhibits enhanced capture efficiency toward both high and low epithelial cell adhesion molecule expression cell lines, and the DNA nanostructure-functionalized chip enables the accurate capture of different phenotypes of CTCs. In addition, the DNA nanoscaffold makes nucleases more accessible to the aptamers to release cells with molecular integrity and outstanding cell viability.

Microgel Single-Cell Culture Arrays on a Microfluidic Chip for Selective Expansion and Recovery of Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are rare and lack definite biomarkers, necessitating new methods for a robust expansion. Here, we developed a microfluidic single-cell culture (SCC) approach for expanding and recovering colorectal CSCs from both cell lines and tumor tissues. By incorporating alginate hydrogels with droplet microfluidics, a high-density microgel array can be formed on a microfluidic chip that allows for single-cell encapsulation and nonadhesive culture. The SCC approach takes advantage of the self-renewal property of stem cells, as only the CSCs can survive in the SCC and form tumorspheres. Consecutive imaging confirmed the formation of single-cell-derived tumorspheres, mainly from a population of small-sized cells. Through on-chip decapsulation of the alginate microgel, ∼6000 live cells can be recovered in a single run, which is sufficient for most biological assays. The recovered cells were verified to have the genetic and phenotypic characteristics of CSCs. Furthermore, multiple CSC-specific targets were identified by comparing the transcriptomics of the CSCs with the primary cancer cells. To summarize, the microgel SCC array offers a label-free approach to obtain sufficient quantities of CSCs and thus is potentially useful for understanding cancer biology and developing personalized CSC-targeting therapies.

Paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells.

Tumor tropism metastasis is a multi-step process that involves interactions between tumor cells and the microenvironment. Due to the limitations of experimental techniques, current studies are not able to gain insight into the dynamic process of such tropism migration. To overcome this issue, we developed a paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells. This co-culture system contains a tumor layer, a recruitment layer, and several invasion layers between these two parts. The tumor and recruitment layers are impregnated with breast cancer cells and lung cells, respectively. Stacking these layers forms a co-culture device that comprises interactions between breast cancer and lung, destacking such a device represents cancer cells at different stages of the migration process. Thus, the paper-supported co-culture system offers the possibility of investigating migration from temporal and spatial aspects. Invasion assays using the co-culture system showed that breast cancer cells induced lung fibroblasts to convert to cancer-associated fibroblasts (CAFs), and the CAFs, in turn, recruited breast cancer cells. During migration, the local invasion of the cancer cells is a collective behavior, while the long-distance migration comes from individual cell behaviors. Breast cancer cells experienced repetitive processes of migration and propagation, accompanied by epithelial-mesenchymal and mesenchymal-epithelial transitions, and changes in stemness and drug resistance. Based on these results, the lung-tropic migration of breast cancer is interpreted as a process of bilateral interaction with the local and host-organ microenvironment. The developed paper-supported co-culture system offers the possibility of dynamically investigating tropism migration under the pre-metastatic niche, thus providing an advantageous tool for studying tumor metastasis.

Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single-Cell Clone-Forming Inhibition Assay.

Screens of cancer stem cells (CSCs)-specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single-cell assay for screening breast cancer stem cell-specific agents. This assay takes advantage of the single-cell clone-forming capability of CSCs, which can be specifically inhibited by CSC-targeting agents. The single-cell assay is performed on a microfluidic chip with an array of 3840 cell-capturing units; the single-cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell-capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long-term suspension culture, only a minority of cells survive and form tumorspheres. The clone-formation rate of MCF-7, MDA-MB-231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone-forming inhibition assay is conducted by exposing the single-cell arrays to a set of anticancer agents. The CSC-targeting agents show complete inhibition of single-cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single-cell assay with the potential to screen CSC-specific agents with high efficiency provides new tools for individualized tumor therapy.