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Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.
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Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Microfluídica/métodos , Análise de Célula Única/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/sangue , Fenótipo , Linhagem Celular Tumoral , Imunoterapia/métodos , Perfilação da Expressão Gênica/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Efficient transfection of therapeutic agents and timely potency testing are two key factors hindering the development of cellular therapy. Here we present a cellular-nanoporation and exosome assessment device, a quantitative platform for nanochannel-based cell electroporation and exosome-based in situ RNA expression analysis. In its application to transfection of anti-miRNAs and/or chemotherapeutics into cells, we have systematically described the differences in RNA expression in secreted exosomes and assessed cellular therapies in real time.
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Exossomos , MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , TransfecçãoRESUMO
Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.
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DNA , MicrofluídicaRESUMO
The genetic heterogeneities in cancer cells pose challenges to achieving precise drug treatment in a widely applicable manner. Most single-cell gene analysis methods rely on cell lysis for gene extraction and identification, showing limited capacity to provide the correlation of genetic properties and real-time cellular behaviors. Here, we report a single living cell analysis nanoplatform that enables interrogating gene properties and drug resistance in millions of single cells. We designed a Domino-probe to identify intracellular target RNAs while releasing 10-fold amplified fluorescence signals. An on-chip addressable microwell-nanopore array was developed for enhanced electro-delivery of the Domino-probe and in situ observation of cell behaviors. The proof-of-concept of the system was validated in primary lung cancer cell samples, revealing the positive-correlation of the ratio of EGFR mutant cells with their drug susceptibilities. This platform provides a high-throughput yet precise tool for exploring the relationship between intracellular genes and cell behaviors at the single-cell level.
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Neoplasias Pulmonares , Análise de Célula Única , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MutaçãoRESUMO
Identifying molecular biomarkers promises to significantly improve the accuracy in cancer diagnosis at its early stage. DNA nanomachines, which are designable and switchable nanostructures made of DNA, show broad potential to detect tumor biomarkers with noninvasive, inexpensive, highly sensitive, and highly specific advantages. This Feature summarizes the recent DNA nanomachine-based platforms for the early detection of cancer biomarkers, both from body fluids and in cells.
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Biomarcadores Tumorais/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Neoplasias/diagnóstico , Técnicas Biossensoriais/métodos , HumanosRESUMO
Stimulated emission depletion (STED) nanoscopy provides subdiffraction resolution while preserving the benefits of fluorescence confocal microscopy in live-cell imaging. However, there are several challenges for multicolor STED nanoscopy, including sophisticated microscopy architectures, fast photobleaching, and cross talk of fluorescent probes. Here, we introduce two types of nanoscale fluorescent semiconducting polymer dots (Pdots) with different emission wavelengths: CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]) Pdots and PDFDP (poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alt-co-{2,5-bis (N,N'-diphenylamino)-1,4-phenylene}]) Pdots, for dual-color STED bioimaging and cellular tracking. Besides bright fluorescence, strong photostability, and easy bioconjugation, these Pdots have large Stokes shifts, which make it possible to share both excitation and depletion beams, thus requiring only a single pair of laser beams for the dual-color STED imaging. Long-term tracking of cellular organelles by the Pdots has been achieved in living cells, and the dynamic interaction of endosomes derived from clathrin-mediated and caveolae-mediated endocytic pathways has been monitored for the first time to propose their interaction models. These results demonstrate the promise of Pdots as excellent probes for live-cell multicolor STED nanoscopy.
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Rastreamento de Células/métodos , Corantes Fluorescentes/uso terapêutico , Polímeros/química , Pontos Quânticos/química , Humanos , LasersRESUMO
The photorelease of bioactive molecules has emerged as a valuable tool in biochemistry. Nevertheless, many important bioactive molecules, such as pyridine derivatives, cannot benefit from currently available organic photoremovable protecting groups (PPGs). We found that the inefficient photorelease of pyridines is attributed to intramolecular photoinduced electron transfer (PET) from PPGs to pyridinium ions. To alleviate PET, we rationally designed a strategy to drive the excited state of PPG from S1 to T1 with a heavy atom, and synthesized a new PPG by substitution of the H atom at the 3-position of 7-dietheylamino-coumarin-4-methyl (DEACM) with Br or I. This resulted in an improved photolytic efficiency of the pyridinium ion by hundreds-fold in aqueous solution. The PPG can be applied to various pyridine derivatives. The successful photorelease of a microtubule inhibitor, indibulin, in living cells was demonstrated for the potential application of this strategy in biochemical research.
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Therapeutic target identification and corresponding drug development is a demanding task for the treatment of lung adenocarcinoma, especially the most malignant proximal-proliferative subtype without druggable protein kinase mutations. Using a cell-SELEX-generated aptamer, we discovered a new tumor driver protein, leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), which is specifically overexpressed in the most lethal subtype of lung adenocarcinoma. Targeted LRPPRC protein knockdown is a promising therapeutic strategy for the undruggable LUAD (lung adenocarcinoma). Nevertheless, LRPPRC is mainly located in mitochondria and degraded by protease. Current protein knockdown approaches, such as proteolysis-targeting chimeras (PROTACs), have limitations in their applications to the proteins degraded through proteasome-independent ways. Here, we designed an aptamer-assisted high-throughput method to screen small molecules that could bind to LRPPRC directly, disrupt the interaction of LRPPRC with its stabilizing chaperon protein, and lead to LRPPRC degradation by mitochondrial protease. The screened compound, gossypolacetic acid (GAA), is an old medicine that can accomplish the new function for targeted LRPPRC knockdown. It showed significant antitumor effects even with the LRPPRC-positive patient-derived tumor xenograft (PDX) model. This work not only extended the application of aptamers to screen small-molecule inhibitors for the undruggable lung cancers, but more importantly provided a new strategy to develop protein knockdown methods beyond the proteasome system.
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Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas de Repetições Ricas em Leucina , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
In vitro, drug assessment holds tremendous potential to success in novel drug development and precision medicine. Traditional techniques for drug assessment, however, face remarkable challenges to achieve high speed, as limited by incubation-based drug delivery (>several hours) and cell viability measurements (>1 d), which significantly compromise the efficacy in clinical trials. In this work, a nano-electroporation-DNA tensioner platform is reported that shortens the time of drug delivery to less than 3 s, and that of cellular mechanical force analysis to 30 min. The platform adopts a nanochannel structure to localize a safe electric field for cell perforation, while enhancing delivery speed by 103 times for intracellular delivery, as compared to molecular diffusion in coculture methods. The platform is further equipped with a DNA tensioner to detect cellular mechanical force for quantifying cell viability after drug treatment. Systematic head-to-head comparison, by analyzing FDA (food and drug administration)-approved drugs (paclitaxel, doxorubicin), demonstrated the platform with high speed, efficiency, and safety, showing a simple yet powerful tool for clinical drug screening and development.
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Sistemas de Liberação de Medicamentos , Eletroporação , Estados Unidos , Eletroporação/métodos , Terapia com Eletroporação , Difusão , DNARESUMO
Analysis of biomarkers in living cells is crucial for deciphering the dynamics of cells as well as for precise diagnosis of diseases. DNA biosensors employ DNA sequences as probes to offer insights into living cells, and drive progress in disease diagnosis and drug development. In this review, we present recent advances in DNA biosensors for detecting biomarkers in living cells. The basic structural components of DNA biosensors and the signal output method are presented. The strategies of DNA biosensors crossing the cell membrane are also described, including coincubation, nanocarriers, and nanoelectroporation techniques. Based on biomarker categorization, we detail recent applications of DNA biosensors for detecting small molecules, RNAs, proteins, and integrated targets in living cells. Furthermore, the future development directions of DNA biosensors are summarized to encourage further research in this growing field.
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Strategies for rapid, effective nucleic acid processing hold tremendous significance to the clinical analysis of circulating tumor DNA (ctDNA), a family of important markers indicating tumorigenesis and metastasis. However, traditional techniques remain challenging to achieve efficient DNA enrichment, further bringing about complicated operation and limited detection sensitivity. Here, we developed an ion concentration polarization microplatform that enabled highly rapid, efficient enrichment and purification of ctDNA from a variety of clinical samples, including serum, urine, and feces. The platform demonstrated efficiently separating and enriching ctDNA within 30 s, with a 100-fold improvement over traditional methods. Integrating an on-chip isothermal amplification module, the platform further achieved 100-fold enhanced sensitivity in ctDNA detection, which significantly eliminated false-negative results in the serum or urine samples due to the low abundance of ctDNA. Such a simple-designed platform offers a user-friendly yet powerful diagnosis technique with a wide applicability, ranging from early tumor diagnosis to infection screening.
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DNA Tumoral Circulante , Neoplasias , Ácidos Nucleicos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , DNA Tumoral Circulante/genética , Carcinogênese , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Fast and accurate detection of Cryptococcus and precise differentiation of its subtypes is of great significance in protecting people from cryptococcal disease and preventing its spread in populations. However, traditional Cryptococcus identification and detection techniques still face significant challenges in achieving high analysis speed as well as high sensitivity. In this work, we report an electric microfluidic biochip. Compared to conventional methods that take several hours or even a day, this chip can detect Cryptococcus within 20 min, and achieve its maximum detection limit within 1 h, with the ability to differentiate between the Cryptococcus neoformans (NEO) and rare Cryptococcus gattii (GAT) efficiently, which accounts for nearly 100%. This device integrated two functional zones of an electroporation lysis (EL) zone for rapid cell lysis (<30 s) and an electrochemical detection (ED) zone for sensitive analysis of the released nucleic acids. The EL zone adopted a design of microelectrode arrays, which obtains a large electric field intensity at the constriction of the microchannel, addressing the safety concerns associated with high-voltage lysis. The device enables a limit of detection (LOD) of 60 pg/mL for NEO and 100 pg/mL for GAT through the modification of nanocomposites and specific probes. In terms of the detection time and sensitivity, the integrated microfluidic biochip demonstrates broad potential in Cryptococcus diagnosis and disease prevention.
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Técnicas Biossensoriais , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Humanos , Criptococose/diagnósticoRESUMO
Precise quantification of low-dose ionizing radiation is of great significance in protecting people from damage caused by clinical radiotherapy or environmental radiation. Traditional techniques for detecting radiation, however, remain extreme challenges to achieve high sensitivity and speed in quantifying radiation dosage. In this work, we report a Cas13a-Microdroplet platform that enables sensitive detection of ultra-low doses of radiation (0.5 Gy vs. 1 Gy traditional) within 1 h. The micro-platform adopts an ideal, specific radiation-sensitive marker, m6A on NCOA4 gene (NCOA4-m6A) that was first reported in our recent work. Microfluidics of the platform generate uniform microdroplets that encapsulate a CRISPR/Cas13a detection system and NCOA4-m6A target from the whole RNA extraction, achieving 10-fold enhancement in sensitivity and significantly reduced limit of detection (LOD). Systematic mouse models and clinical patient samples demonstrated its superior sensitivity and LOD (0.5 Gy) than traditional qPCR, which show wide potentials in radiation tracking and damage protection.
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Técnicas Biossensoriais , Animais , Camundongos , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Limite de Detecção , Doses de Radiação , Fatores de Transcrição , Coativadores de Receptor NuclearRESUMO
Enhancers involved in the upregulation of multiple oncogenes play a fundamental role in tumorigenesis and immortalization. Exploring the activity of enhancers in living cells has emerged as a critical path to a deep understanding of cancer properties, further providing important clues to targeted therapy. However, identifying enhancer activity in living cells is challenging due to the double biological barriers of a cell cytoplasmic membrane and a nuclear membrane, limiting the sensitivity and responsiveness of conventional probing methods. In this work, we developed a nanoelectroporation-probing (NP) platform, which enables intranuclear probe delivery for sensitive interrogation of enhancer activity in living cells. The nanoelectroporation biochip achieved highly focused perforation of the cell cytoplasmic membrane and brought about additional driving force to expedite the delivery of probes into the nucleus. The probes targeting enhancer activity (named "PH probe") are programmed with a cyclic amplification strategy and enable an increase in the fluorescence signals over 100-fold within 1 h. The platform was leveraged to detect the activity of CCAT1 enhancers (CCAT1, colon cancer-associated transcript-1, a long noncoding RNA that functions in tumor invasion and metastasis) in cell samples from clinical lung cancer patients, as well as reveal the heterogeneity of enhancers among different patients. The observations may extend the linkages between enhancers and cancer cells while validating the robustness and reliability of the platform for the assay of enhancer activity. This platform will be a promising toolbox with wide applicable potential for the intranuclear study of living cells.
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Neoplasias Pulmonares , Humanos , Reprodutibilidade dos TestesRESUMO
Corona Virus Disease 2019 (COVID-19) has developed into a global pandemic in the last two years, causing significant impacts on our daily life in many countries. Rapid and accurate detection of COVID-19 is of great importance to both treatments and pandemic management. Till now, a variety of point-of-care testing (POCT) approaches devices, including nucleic acid-based test and immunological detection, have been developed and some of them has been rapidly ruled out for clinical diagnosis of COVID-19 due to the requirement of mass testing. In this review, we provide a summary and commentary on the methods and biomedical devices innovated or renovated for the quick and early diagnosis of COVID-19. In particular, some of micro and nano devices with miniaturized structures, showing outstanding analytical performances such as ultra-sensitivity, rapidness, accuracy and low cost, are discussed in this paper. We also provide our insights on the further implementation of biomedical devices using advanced micro and nano technologies to meet the demand of point-of-care diagnosis and home testing to facilitate pandemic management. In general, our paper provides a comprehensive overview of the latest advances on the POCT device for diagnosis of COVID-19, which may provide insightful knowledge for researcher to further develop novel diagnostic technologies for rapid and on-site detection of pathogens including SARS-CoV-2.
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Probing nuclear protein expression while correlating cellular behavior is crucial for deciphering underlying causes of cellular disorders, such as tumor metastasis. Despite efforts to access nuclear proteins by trafficking the double barriers of cell membrane and nuclear membrane, they mostly fall short of the capacity for analyzing various proteins in different cells. Herein, we introduce a Companion-Probe & Race (CPR) platform that enables interrogating nuclear proteins in living cells, while guiding and tracking cellular behaviors (e.g., migration) in real time. The Companion-Probe consists of two polypeptide complexes that were structured with nuclear localization signal (NLS) for entering nucleus, recognition polypeptide for targeting different sites of nuclear proteins, and fragments of green fluorescent protein (GFP) that can recover a whole fluorescent GFP once the two polypeptide complexes combine with a same target protein. The two polypeptide complexes were expressed by two plasmids (named "probe plasmids") that were uniformly and efficiently delivered into cells by nano-electroporation (NEP), a high-performance delivery method for cell focal-poration and accelerated intracellular delivery. To track cell migration, multiple radial microchannels were designed with micro-landmarks on the platform to serve as addressable runways for cells. The proof-of-concept of CPR platform was validated with clinical primary cells that indicated the positive-correlation between nuclear protein murine double minute 2 (MDM2) expression level and cell migration velocity. This platform shows great promises to interrogate nuclear proteins in live cells, and to decode their roles in determining cellular behaviors on a chip.
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Técnicas Biossensoriais , Proteínas Nucleares , Animais , Núcleo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies µL-1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Ouro , Reação em Cadeia da Polimerase Multiplex , MutaçãoRESUMO
The occurrence and development of many diseases are accompanied and sometimes dictated by the destruction of biomechanical homeostasis. For instance, cancer cells and normal cells show different cellular mechanical forces phenotypes, as the proliferation and invasion ability of cancer cells is often related to the changes in mechanical force in the tumor. With single cell analysis, variations in mechanics within a cell population can be detected and analyzed, opening new dimensions in the study of cancer. Nanosensor design for interrogation of cell mechanics is an interdisciplinary area bridging over cell biology, mechanics, and micro/nanotechnology. In this tutorial review, we give insight into the background and technical innovation of currently available methods for mechanical analysis of cells. First, we discuss the mechanism of mechanical changes in the development and progression of cancer that shows the feasibility of mechanical sensors in cancer cell detection. Next, we summarize the principle, progress, and essential problems of common technologies for cell force measurement, including single molecule force spectroscopy and elastic substrate-sensors. Following that, we discuss novel micro and nano-scale mechanical sensors and their applications in single cell level biological analysis. At last, we elaborate on the remaining issues and trends of the cellular mechanical sensors.
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Técnicas Biossensoriais , Fenômenos Mecânicos , Nanotecnologia , Análise de Célula Única , Estresse MecânicoRESUMO
Sensitive detection of SARS-CoV-2 is of great importance for inhibiting the current pandemic of COVID-19. Here, we report a simple yet efficient platform integrating a portable and low-cost custom-made detector and a novel microwell array biochip for rapid and accurate detection of SARS-CoV-2. The instrument exhibits expedited amplification speed that enables colorimetric read-out within 25 minutes. A polymeric chip with a laser-engraved microwell array was developed to process the reaction between the primers and the respiratory swab RNA extracts, based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). To achieve clinically acceptable performance, we synthesized a group of six primers to identify the conserved regions of the ORF1ab gene of SARS-CoV-2. Clinical trials were conducted with 87 PCR-positive and 43 PCR-negative patient samples. The platform demonstrated both high sensitivity (95.40%) and high specificity (95.35%), showing potentials for rapid and user-friendly diagnosis of COVID-19 among many other infectious pathogens.
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Bio-sourced hydrogels are attractive materials for diagnosing, repairing and improving the function of human tissues and organs. However, their mechanical strength decreases with an increase in water content. Furthermore, it is challenging to mold these hydrogels with high precision, which significantly limits their applications. Herein, we modified a biocompatible and biodegradable material, hyaluronic acid, with methacrylic anhydride and then cured it with a four-arm star structure cross-linking agent under ultraviolet light. The hyaluronic acid hydrogel was finally cured within 15 s with an adjustable cross-linking degree. The results demonstrated that the developed gel maintained good mechanical strength with a water content of 90%, while achieving micropatterns at a precision of 20 µm. The biological experiments showed that it could effectively promote the release of vascular endothelial growth factor (VEGF), which contributed to promoting cell growth, and has favorable biocompatibility. Overall, this hyaluronic acid hydrogel is a promising biomedical material with high forming accuracy, excellent mechanical properties, and favorable biocompatibility, which indicate its potential value in a variety of tissue engineering and biomedical applications.