A Microfluidic Chip-Based Automated System for Whole-Course Monitoring the Drug Responses of Organoids.

Tumor patients-derived organoids, as a promising preclinical prediction model, have been utilized to evaluate ex vivo drug responses for formulating optimal therapeutic strategies. Detecting adenosine triphosphate (ATP) has been widely used in existing organoid-based drug response tests. However, all commercial ATP detection kits containing the cell lysis procedure can only be applied for single time point ATP detection, resulting in the neglect of dynamic ATP variations in living cells. Meanwhile, due to the limited number of viable organoids from a single patient, it is impractical to exhaustively test all potential time points in search of optimal ones. In this work, a multifunctional microfluidic chip was developed to perform all procedures of organoid-based drug response tests, including establishment, culturing, drug treatment, and ATP monitoring of organoids. An ATP sensor was developed to facilitate the first successful attempt on whole-course monitoring the growth status of fragile organoids. To realize a clinically applicable automatic system for the drug testing of lung cancer, a microfluidic chip based automated system was developed to perform entire organoid-based drug response test, bridging the gap between laboratorial manipulation and clinical practices, as it outperformed previous methods by improving data repeatability, eliminating human error/sample loss, and more importantly, providing a more accurate and comprehensive evaluation of drug effects.

[Application and Research Progress of Lung Cancer Organoid in Precision Medicine 
for Lung Cancer].

The continuous advancement of molecular detection technology has greatly propelled the development of precision medicine for lung cancer. However, tumor heterogeneity is closely associated with tumor metastasis, recurrence, and drug resistance. Additionally, different lung cancer patients with the same genetic mutation may exhibit varying treatment responses to different therapeutic strategies. Therefore, the development of modern precision medicine urgently requires the precise formulation of personalized treatment strategies through personalized tumor models. Lung cancer organoid (LCO) can highly simulate the biological characteristics of tumor in vivo, facilitating the application of innovative drugs such as antibody-drug conjugate in precision medicine for lung cancer. With the development of co-culture model of LCO with tumor microenvironment and tissue engineering technology such as microfluidic chip, LCO can better preserve the biological characteristics and functions of tumor tissue, further improving high-throughput and automated drug sensitivity experiment. In this review, we combine the latest research progress to summarize the application progress and challenges of LCO in precision medicine for lung cancer.
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A flexible electrode Array for genetic transfection of different layers of the retina by electroporation.

Electroporation (in which the permeability of a cell membrane is increased transiently by exposure to an appropriate electric field) has exhibited great potential of becoming an alternative to adeno-associated virus (AAV)-based retina gene delivery. Electroporation eliminates the safety concerns of employing exogenous viruses and exceeds the limit of AAV cargo size. Unfortunately, several concerns (e.g., relatively high electroporation voltage, poor surgical operability and a lack of spatial selectivity of retina tissue) have prevented electroporation from being approved for clinical application (or even clinical trials). In this study, a flexible micro-electrode array for retina electroporation (FERE) was developed for retina electroporation. A suitably shaped flexible substrate and well-placed micro-electrodes were designed to adapt to the retina curvature and generate an evenly distributed electric field on the retina with a significantly reduced electroporation voltage of 5 V. The FERE provided (for the first time) a capability of controlled gene delivery to the different structural layers of retina tissue by precise control of the distribution of the electrical field. After ensuring the surgical operability of the FERE on rabbit eyeballs, the FERE was verified to be capable of transfecting different layers of retina tissue with satisfactory efficiency and minimum damage. Our method bridges the technical gap between laboratory validation and clinical use of retina electroporation.

A dual-functional microfluidic chip for guiding personalized lung cancer medicine: combining EGFR mutation detection and organoid-based drug response test.

Many efforts have been paid to advance the effectiveness of personalized medicine for lung cancer patients. Sequencing-based molecular diagnosis of EGFR mutations has been widely used to guide the selection of anti-lung-cancer drugs. Organoid-based assays have also been developed to ex vivo test individual responses to anti-lung-cancer drugs. After addressing several technical difficulties, a new combined strategy, in which anti-cancer medicines are first selected based on molecular diagnosis and then ex vivo tested on organoids, has been realized in a single dual-functional microfluidic chip. A DNA-based nanoruler has been developed to detect the existence of EGFR mutations and shrink the detection period from weeks to hours, compared with sequencing. The employment of the DNA-based nanoruler creates a possibility to purposively test anti-cancer drugs, either EGFR-TKIs or chemotherapy drugs, not both, on limited amounts of organoids. Moreover, a DNA-based nanosensor has been developed to recognize intracellular ATP variation without harming cell viability, realizing in situ monitoring of the whole course growth status of organoids for on-chip drug response test. The dual-functional microfluidic chip was validated by both cell lines and clinical samples from lung cancer patients. Furthermore, based on the dual-functional microfluidic chip, a fully automated system has been developed to span the divide between experimental procedures and therapeutic approaches. This study constitutes a novel way of combining EGFR mutation detection and organoid-based drug response test on an individual patient for guiding personalized lung cancer medicine.

Rapid determination of the presence of EGFR mutations with DNA-based nanocalipers.

Selecting 1st-line treatment for lung cancer is currently a binary choice, either chemotherapy or targeted medicine, depending on whether EGFR mutations exist. Next-generation sequencing is fully capable of accurately identifying EGFR mutations and guiding the usage of tyrosine kinase inhibitors, but it is highly expensive. Moreover, as the sequencing is not helpful for patients with wild-type EGFR, the long wait for sequencing may delay the chemotherapy and correspondingly increase the risks of cancer progression. To address this issue, a new method for rapidly determining the presence of EGFR mutations is developed in this study. A series of DNA origami-engineered nanocalipers are designed and constructed to determine the EGFR spatial distribution of either mutated EGFR or wild-type EGFR lung cancer cells. The experimental results on cancer cell lines and 9 clinical tissue samples show that compared with wild-type EGFR cells, mutated EGFR cells have narrower EGFR spacing. Hence, the DNA nanocalipers are demonstrated to be capable of determining the presence of EGFR mutations and shrinking the detection period from weeks to hours, compared with sequencing. For determining EGFR mutation status in 9 clinical samples, DNA nanocalipers show 100% consistency with next-generation sequencing.

Real-time monitoring ATP variation in human cancer organoids for a long term by DNA-based nanosensor.

Cancer organoids have become promising tools for predicting drug responses on many different types of cancer. Detecting the adenosine triphosphate (ATP) has currently been considered as a decisive test to profile the growth status and drug responses of organoids. ATP profiling using commercial ATP detection kits, which involve cell lysis, can be performed at a single time spot, causing a clinical dilemma of selecting the optimal time spot to adopt diverse cancer types and patients. This study provides a feasible solution to this dilemma by developing a DNA-based ATP nanosensor to realize real-time ATP monitoring in organoids for a long term. The employment of DNA materials ensures high biocompatibility and low cytotoxicity, which are crucial for fragile organoids; The usage of tetrahedral DNA framework ensures cell permeability and intracellular ATP detection; The introduction of ATP-mediated molecular replacement ensures the high sensitivity and selectivity of ATP recognition. These features result in the first successful attempt on real-time monitoring ATP in organoids for up to 26 days and gaining growth status curves for the whole duration of a drug sensitivity test on human lung cancer organoids.

Mechanisms, Techniques and Devices of Airborne Virus Detection: A Review.

Airborne viruses, such as COVID-19, cause pandemics all over the world. Virus-containing particles produced by infected individuals are suspended in the air for extended periods, actually resulting in viral aerosols and the spread of infectious diseases. Aerosol collection and detection devices are essential for limiting the spread of airborne virus diseases. This review provides an overview of the primary mechanisms and enhancement techniques for collecting and detecting airborne viruses. Indoor virus detection strategies for scenarios with varying ventilations are also summarized based on the excellent performance of existing advanced comprehensive devices. This review provides guidance for the development of future aerosol detection devices and aids in the control of airborne transmission diseases, such as COVID-19, influenza and other airborne transmission viruses.

Dual-function microneedle array for efficient photodynamic therapy with transdermal co-delivered light and photosensitizers.

Photodynamic therapy (PDT), as a globally accepted method for treating different forms of skin or mucosal disorders, requires efficient co-delivery of photosensitizers and corresponding therapeutic light. The adverse effects of intravenous injection of photosensitizers have been reduced by the development of microneedle arrays for transdermal local photosensitizer delivery. However, the drawbacks of the only available therapeutic light delivery method at the moment, which is directly applying light to the skin surface, are yet to be improved. This study presents a new strategy in which therapeutic light and photosensitizer were transdermally co-delivered into local tissues. A flexible dual-function microneedle array (DfMNA) which contains 400 microneedles was developed. Each microneedle consists of a dissolvable needle tip (140 µm in height) for delivering the photosensitizer and a transparent needle body (660 µm in height) for guiding therapeutic light. Using port-wine stains, which is a frequently occurring skin disorder caused by vascular malformation, as a model disease, the effectiveness of DfMNA mediated PDT has been verified on mice. Compared with the standard operation procedure of clinical PDT, the DfMNA decreases the amount of photosensitizer from 300 µg to 0.5 µg and reduces therapeutic light irradiance from 100 mW cm-2 to 60 mW cm-2 while realizing better treatment effects. As a result, the skin damage and the burden on the metabolic system have been alleviated. The DfMNA has a remarkably reduced photosensitizer amount and, for the first time, realized transdermal delivery of therapeutic light for PDT, thus avoiding the disadvantages of existing PDT methodologies.

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics.

Single-cell nucleic acid analysis aims at discovering the genetic differences between individual cells which is well known as the cellular heterogeneity. This technology facilitates cancer diagnosis, stem cell research, immune system analysis, and other life science applications. The conventional platforms for single-cell nucleic acid analysis more rely on manual operation or bulky devices. Recently, the emerging microfluidic technology has provided a perfect platform for single-cell nucleic acid analysis with the characteristic of accurate and automatic single-cell manipulation. In this review, we briefly summarized the procedure of single-cell nucleic acid analysis including single-cell isolation, single-cell lysis, nucleic acid amplification, and genetic analysis. And then, three representative microfluidic platforms for single-cell nucleic acid analysis are concluded as valve-, microwell-, and droplet-based platforms. Furthermore, we described the state-of-the-art integrated single-cell nucleic acid analysis systems based on the three platforms. Finally, the future development and challenges of microfluidics-based single-cell nucleic acid analysis are discussed as well.

A microfluidic cytometer for white blood cell analysis.

Despite the wide use of cytometry for white blood cell classification, the performance of traditional cytometers in point-of-care testing remains to be improved. Microfluidic techniques have been shown with considerable potentials in the development of portable devices. Here we present a prototype of microfluidic cytometer which integrates a three-dimensional hydrodynamic focusing system and an on-chip optical system to count and classify white blood cells. By adjusting the flow speed of sheath flow and sample flow, the blood cells can be horizontally and vertically focused in the center of microchannel. Optical fibers and on-chip microlens are embedded for the excitation and detection of single-cell. The microfluidic chip was validated by classifying white blood cells from clinical blood samples.

A Microfluidic Chip for Screening and Sequencing of Monoclonal Antibody at a Single-Cell Level.

The pairing of heavy and light chains of an antibody decides the specificity of monoclonal antibodies (mAbs). Acquisition of the genes encoding variable regions of paired heavy and light chains (VH:VL) is crucial, but it is a labor- and cost-intensive process in traditional methods. The emerging microfluidic chips have brought us to a portal of directly acquiring natively paired VH:VL genes by sequencing single target cells. This study presents a novel method in which all processing steps for acquiring natively paired VH:VL genes from single cells are finished in a single microfluidic chip, not multiple discrete devices. The microfluidic chip performs single-cell trapping/in situ fluorescence examination of antibody specificity/cell lysis/gene amplification all at a single-cell level. By a proof-of-concept validation of efficiently acquiring paired VH:VL genes of anti-CD45 mAbs from single hybridoma cells, the microfluidic chip has been proved capable of trapping/screening/lysing single antibody-secreting cells and performing an on-chip reverse transcription-polymerase chain reaction. The presented method has realized remarkably improved cell loss/human labor/time cost, and more importantly, determinacy of native VH:VL gene pairing, which is one of the most decisive factors of effectiveness for antibody discovery.

A Microfluidic Chip for Efficient Circulating Tumor Cells Enrichment, Screening, and Single-Cell RNA Sequencing.

Single-cell RNA sequencing on circulating tumor cells (CTCs) proves useful to study mechanisms of tumor heterogeneity, metastasis, and drug resistance. Currently, single-cell RNA sequencing of CTCs usually takes three prerequisite steps: enrichment of CTCs from whole blood, characterization of captured cells by immunostaining and microscopic imaging, and single-cell isolation through micromanipulation. However, multiple pipetting and transferring steps can easily cause the loss of rare CTCs. To address this issue, a novel integrated microfluidic chip for sequential enrichment, isolation, and characterization of CTCs at single-cell level, is developed. And, single CTC lysis is achieved on the same chip. The microfluidic chip includes functions of blood clot filtration, single-cell isolation, identification, and target single-cell lysate collection. By spiking tumor cells into whole blood, it is validated that this microfluidic chip can effectively conduct single-cell CTCs RNA sequencing. The approach lays a solid foundation for the analysis of RNA expression profiling of single-cell CTCs.

A microfluidic chip for screening high-producing hybridomas at single cell level.

Hybridomas are a commonly used, or even the only option, for laboratory study and pilot production of monoclonal antibodies (mAbs), which are crucial for both targeted therapy and biomedical study. A long-term culture of hybridomas will inevitably induce a heterogenization of the whole hybridoma population, resulting in a continuous growth of non-producing hybridomas. To overcome the limits of existing methods of screening heterogeneous hybridomas, in which the whole multi-round screening process is performed in multi-well plates or other discrete modules, this study presents a novel method in which all processing steps of a multi-round hybridoma screening are finished in a single microfluidic chip. This microfluidic chip comprehensively performs hybridoma trapping/proliferating/transferring and fluorescent identification of protein-antibody binding at single cell level. By performing a two-round screening of anti-CD45 mAb secreting hybridomas, the novel microfluidic chip was proved capable of screening several single high-producing hybridomas with minimum cell loss/human labor/time cost, and more importantly, enhanced accuracy and definite monoclonality, which is one of the most important properties of mAb production.

Device for whole genome sequencing single circulating tumor cells from whole blood.

Whole-genome sequencing on circulating tumor cells (CTCs) at the single cell level has recently been found helpful for precision medicine, as the oncogenic profiles of single CTCs are useful for discovering oncogenic mutation heterogeneities and guiding/adjusting cancer treatment. To overcome the limits of existing methods of single CTC sequencing, in which CTC enrichment, identification and gene amplification are performed by discrete modules, this study presents a novel method in which all processing steps from blood sample collection to preparation of gene amplification products for sequencers are finished in a single microfluidic chip. This microfluidic chip comprehensively performs blood filtering, CTC enrichment, CTC identification/isolation, CTC lysis and whole genome amplification (WGA) at the single cell level. By sequencing single CTCs from clinical blood samples with pointing key driver and drug-resistance mutations, the novel microfluidic chip was validated to be capable of genetically profiling single CTCs with minimum cell loss/human labor, and more importantly, high accuracy and repeatability, which are crucial factors for promoting clinical application of single CTC sequencing.

An oligonucleotide synthesizer based on a microreactor chip and an inkjet printer.

Synthetic oligonucleotides (oligos) are important tools in the fields of molecular biology and genetic engineering. For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo. This study reports a microreactor chip for oligo synthesis. By incorporating silica beads in the microreactors, the surface area of the solid substrate for oligo synthesis increases significantly in each microreactor. These beads are fixed in the microreactors to withstand the flushing step in oligo synthesis. Compared to conventional synthesis methods, this design is able to avoid protocols to hold the beads and integrate more microreactors on a chip. An inkjet printer is utilized to deliver chemical reagents in the microreactors. To evaluate the feasibility of oligo synthesis using this proof-of-concept synthesizer, an oligo with six nucleotide units is successfully synthesized.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip.

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.

Combined peripheral natural killer cell and circulating tumor cell enumeration enhance prognostic efficiency in patients with metastatic triple-negative breast cancer.

OBJECTIVE: Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis. Circulating tumor cells (CTCs) are a promising predictor for breast cancer prognoses but their reliability regarding progression-free survival (PFS) is controversial. We aim to verify their predictive value in TNBC. METHODS: In present prospective cohort study, we used the Pep@MNPs method to enumerate CTCs in baseline blood samples from 75 patients with TNBC (taken at inclusion in this study) and analyzed correlations between CTC numbers and outcomes and other clinical parameters. RESULTS: Median PFS was 6.0 (range: 1.0-25.0) months for the entire cohort, in whom we found no correlations between baseline CTC status and initial tumor stage (P=0.167), tumor grade (P=0.783) or histological type (P=0.084). However, among those getting first-line treatment, baseline CTC status was positively correlated with ratio of peripheral natural killer (NK) cells (P=0.032), presence of lung metastasis (P=0.034) and number of visceral metastatic site (P=0.037). Baseline CTC status was predictive for PFS in first-line TNBC (P=0.033), but not for the cohort as a whole (P=0.118). This prognostic limitation of CTC could be ameliorated by combining CTC and NK cell enumeration (P=0.049). CONCLUSIONS: Baseline CTC status was predictive of lung metastasis, peripheral NK cell ratio and PFS in TNBC patients undergoing first-line treatment. We have developed a combined CTC-NK enumeration strategy that allows us to predict PFS in TNBC without any preconditions.

Quantitative Proteomic Analysis of Cell Responses to Electroporation, a Classical Gene Delivery Approach.

Electroporation, as an established nonviral technology for breaching cell membrane, has been accepted for the delivery of nucleic acids. Despite satisfactory delivery efficiencies have been achieved on multiple cell kinds by simply exhausting all possible electrical parameters, electroporation is still inefficient, or even invalid, for various kinds of cells. This is largely due to the lack of comprehensive understanding of cell responses to electrical stimulation at biological aspect. Moreover, a systematically investigation of protein variation of electroporated cells is also required for biosafety evaluation before clinically applying electroporation. By employing quantitative proteomic analysis, the biological mechanism of electroporation is explored from the molecular level. The results reveal that electrical stimulations widely influence many biological processes including nucleic acid stabilization, protein synthesis, cytoskeleton dynamic, inflammation, and cell apoptosis. It is found that several antivirus-related processes appeared in the enrichment results. Moreover, SAMD9, a broad spectrum antiviral and antitumor factor, is dramatically downregulated on easy-to-transfect cells while electroporation can not alter SAMD9 expression on hard-to-transfect cells, hinting that electroporation, a pure physical treatment, can induce antivirus-like defensive responses and the altering of SAMD9 can be used to predict the effectiveness of electroporation on transfecting specific kinds of cells.

Efficient delivery of nucleic acid molecules into skin by combined use of microneedle roller and flexible interdigitated electroporation array.

Rationale: Delivery of nucleic acid molecules into skin remains a main obstacle for various types of gene therapy or vaccine applications. Here we propose a novel electroporation approach via combined use of a microneedle roller and a flexible interdigitated electroporation array (FIEA) for efficient delivery of DNA and siRNA into mouse skin. Methods: Using micromachining technology, closely spaced gold electrodes were made on a pliable parylene substrate to form a patch-like electroporation array, which enabled close surface contact between the skin and electrodes. Pre-penetration of the skin with a microneedle roller resulted in the formation of microchannels in the skin, which played a role as liquid electrodes in the skin and provided a uniform and deep electric field in the tissue when pulse stimulation was applied by FIEA. Results: Using this proposed method, gene (RFP) expression and siRNA transfection were successfully achieved in normal mice skin. Anti-SCD1 siRNA electroporated via this method mediated significant gene silencing in the skin. Moreover, electroporation assisted by the microneedle roller showed significant advantages over treatment with FIEA alone. This allowed nucleic acid transportation at low voltage, with ideal safety outcomes. Principal conclusions: Hence, the proposed electroporation approach in this study constitutes a novel way for delivering siRNA and DNA, and even other nucleic acid molecules, to mouse skin in vivo, potentially supporting clinical application in the treatment of skin diseases or intradermal/subcutaneous vaccination.

Generation of a monoclonal antibody recognizing the heavily glycosylated CD45 protein and its application on identifying circulating tumor cells.

Here, we provide direct evidence that using recombinant proteins expressed in eukaryotic cells as antigen is a practical way to generate monoclonal antibodies (mAbs) against heavily glycosylated proteins. Heavily glycosylated proteins are typically difficult targets for mAb generation, being limited by unsatisfactory affinity and low specificity. Using the heavily glycosylated CD45 protein as an example, we demonstrate the entire process of expressing the protein in eukaryotic cells and using it as an antigen to generate CD45-targeting mAbs in mice. The mAbs generated showed robust affinity and specificity, which are crucial factors for differentiate circulating tumor cells from white blood cells in human breast cancer patient samples. Only 1 cell fusion and 2 cyclic sub-cloning steps were necessary before mAbs with satisfactory performance were obtained.