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Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.
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In this paper we demonstrate how the use of frequencies ranging from 50 kHz to 5 GHz in the analysis of cells by electrorotation can open the path to the identification of differences not detectable by conventional set-ups. Earlier works usually reported electrorotation devices operating below 20 MHz, limiting the response obtained to properties associated with the cell membrane. Those devices are thus unable to resolve the physiological properties in the cytoplasm. We used microwave-based technology to extend the frequency operation to 5 GHz. At high frequencies (from tens of MHz to GHz), the electromagnetic signal passes through the membrane and allows probing the cytoplasm. This enables several applications, such as cell classification, and viability analysis. Additionally, the use of conventional microfabrication techniques reduces the cost and complexity of analysis, compared to other non-invasive methods. We demonstrated the potential of this set-up by identifying two different populations of T-lymphocytes not distinguishable through visual assessment. We also assessed the effect of calcein on cell cytoplasmic properties and used it as a controlled experiment to demonstrate the possibility of this method to detect changes happening predominantly in the cytoplasm.
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Conductividad Eléctrica , Citoplasma , Membrana CelularRESUMEN
In proteomics, the need to precisely examine the protein compounds of small samples, requires sensitive analytical methods which can separate and enrich compounds with high precision. Current techniques require a minimal analysis time to obtain satisfactory compound separation where longer analysis time means better separation of compounds. But, molecular diffusion will create broadening of the separated compound bands over time, increasing the peak width, and thus reducing the resolution and the enrichment. Electric field gradient focusing (EFGF) is a separation technique, in which proteins are simultaneously separated and enriched by balancing a gradient electrostatic force with a constant hydrodynamic drag force. Because of this balance, analytes are continuously pushed back to their focusing point, limiting the time-dependent peak broadening due to molecular diffusion. Current EFGF techniques are however still suffering from peak broadening because of flow-profile inhomogeneities. In this paper, we propose to use AC electro-osmotic flow (AC EOF) to create a homogeneous flow in EFGF. The interference between the electric field gradient and the AC EOF was thoroughly analysed and the concept was validated using numerical simulations. The results show that a plug flow is obtained on top of a small, distorted boundary layer. While applying different DC electric fields in the electrolyte, a constant flow velocity can be obtained by including a DC offset to the electrodes generating the AC EOF. The plug flow is then maintained over the whole separation channel length, while an electric field gradient is applied. This way, the flow-induced contribution to peak broadening can be minimized in EFGF devices. By modelling the separation of green fluorescent protein (GFP) and R-Phycoerythrin (R-PE), it was shown that the peak width of separated compounds can be reduced and that the separation resolution can be improved, compared to current EFGF methods.
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Electricidad , Proteínas Fluorescentes Verdes , TiempoRESUMEN
The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.
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Técnicas Biosensibles , COVID-19 , COVID-19/diagnóstico , Humanos , Estudios Longitudinales , ARN Viral/análisis , Aerosoles y Gotitas Respiratorias , SARS-CoV-2 , SilicioRESUMEN
BACKGROUND: Extrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine. METHODS: Here, we report the discovery and analysis of urinary cell-free eccDNAs (ucf-eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle-Seq. RESULTS: Millions of unique ucf-eccDNAs were identified and comprehensively characterised. The ucf-eccDNAs are GC-rich. Most ucf-eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf-eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein-coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf-eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf-eccDNA. CONCLUSIONS: This work discovers and provides the first deep characterisation of ucf-eccDNAs and suggests ucf-eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.
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ADN Circular , Insuficiencia Renal Crónica , Biomarcadores , ADN , ADN Circular/genética , Femenino , Genómica , Humanos , Masculino , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/genéticaRESUMEN
Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.
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Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Circular/genética , ARN Guía de Kinetoplastida/genética , Alelos , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , ADN Circular/metabolismo , Edición Génica/métodos , Células HEK293 , Haplotipos , Células Hep G2 , Humanos , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Integration of microelectronics with microfluidics enables sophisticated lab-on-a-chip devices for sensing and actuation. In this paper, we investigate a novel method for in-situ microfluidics fabrication and packaging on wafer level. Two novel photo-patternable adhesive polymers were tested and compared, PA-S500H and DXL-009. The microfluidics fabrication method employs photo lithographical patterning of spin coated polymer films of PA or DXL and direct bonding of formed microfluidics to a top glass cover using die-to-wafer level bonding. These new adhesive materials remove the need for additional gluing layers. With this approach, we fabricated disposable microfluidic flow cytometers and evaluated the performance of those materials in the context of this application. DXL-009 exhibits lower autofluorescence compared to PA-S500H which improves detection sensitivity of fluorescently stained cells. Results obtained from the cytotoxicity test reveals that both materials are biocompatible. The functionality of these materials was demonstrated by detection of immunostained monocytes in microfluidic flow cytometers. The flexible, fully CMOS compatible fabrication process of these photo-patternable adhesive materials will simplify prototyping and mass manufacturing of sophisticated microfluidic devices with integrated microelectronics.
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Adhesivos/química , Citometría de Flujo/instrumentación , Dispositivos Laboratorio en un Chip , Animales , Fibroblastos , Citometría de Flujo/métodos , Humanos , Ensayo de Materiales , Ratones , Polímeros/química , Relación Señal-RuidoRESUMEN
Safe, high-rate and cost-effective cell sorting is important for clinical cell isolation. However, commercial fluorescence-activated cell sorters (FACS) are expensive and prone to aerosol-induced sample contamination. Here we report a microfluidic cell sorter allowing high rate and fully enclosed cell sorting. The sorter chip consists of an array of micro heating hotspots. Pulsed resistive heating in the hotspots produces numerous micro vapor bubbles with short duration, which gives rise to a rapid jet flow for cell sorting. With this method, we demonstrated high sorting rate comparable to commercial FACS and the significant enrichment of rare cancer cells. This vapor bubble based cell sorting method can be a powerful tool for contamination-free and affordable clinical cell sorting such as circulating tumor cell isolation and cancer cell therapy.
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Citometría de Flujo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Línea Celular Tumoral , Diseño de Equipo , Citometría de Flujo/métodos , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Genetic engineering and screening of large number of cells or populations is a crucial bottleneck in today's systems biology and applied (micro)biology. Instead of using standard methods in bottles, flasks or 96-well plates, scientists are increasingly relying on high-throughput strategies that miniaturize their experiments to the nanoliter and picoliter scale and the single-cell level. In this review, we summarize different high-throughput system-wide genome engineering and screening strategies for microbes. More specifically, we will emphasize the use of multiplex automated genome evolution (MAGE) and CRISPR/Cas systems for high-throughput genome engineering and the application of (lab-on-chip) nanoreactors for high-throughput single-cell or population screening.
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Bacterias/genética , Biotecnología/métodos , Ingeniería Genética/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Sistemas CRISPR-Cas/genética , MicrofluídicaRESUMEN
Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 µm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.
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Adenocarcinoma/patología , Neoplasias de la Mama/patología , Ensayos de Migración Celular/instrumentación , Dispositivos Laboratorio en un Chip , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Microambiente Tumoral , Adenocarcinoma/diagnóstico , Neoplasias de la Mama/diagnóstico , Línea Celular Tumoral , Movimiento Celular , Diseño de Equipo , Femenino , Ensayos Analíticos de Alto Rendimiento , Holografía , Humanos , Microscopía de Contraste de Fase , Pruebas en el Punto de Atención , Impresión Tridimensional , Reproducibilidad de los ResultadosRESUMEN
A compelling clinical need exists for inexpensive, portable haematology analyzers that can be utilized at the point-of-care in emergency settings or in resource-limited settings. Development of a label-free, microfluidic blood analysis platform is the first step towards such a miniaturized, cost-effective system. Here we assemble a compact lens-free in-line holographic microscope and employ it to image blood cells flowing in a microfluidic chip, using a high-speed camera and stroboscopic illumination. Numerical reconstruction of the captured holograms allows classification of unlabeled leukocytes into three main subtypes: lymphocytes, monocytes and granulocytes. A scale-space recognition analysis to evaluate cellular size and internal complexity is also developed and used to build a 3-part leukocyte differential. The lens-free image-based classification is compared to the 3-part white blood cell differential generated by using a conventional analyzer on the same blood sample and is found to be in good agreement with it.
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Citometría de Flujo/instrumentación , Dispositivos Laboratorio en un Chip , Leucocitos/citología , Técnicas Analíticas Microfluídicas , Voluntarios Sanos , Humanos , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
We present a novel opto-magnetic system for the fast and sensitive detection of nucleic acids. The system is based on a lens-free imaging approach resulting in a compact and cheap optical readout of surface hybridized DNA fragments. In our system magnetic particles are attracted towards the detection surface thereby completing the labeling step in less than 1 min. An optimized surface functionalization combined with magnetic manipulation was used to remove all nonspecifically bound magnetic particles from the detection surface. A lens-free image of the specifically bound magnetic particles on the detection surface was recorded by a CMOS imager. This recorded interference pattern was reconstructed in software, to represent the particle image at the focal distance, using little computational power. As a result we were able to detect DNA concentrations down to 10 pM with single particle sensitivity. The possibility of integrated sample preparation by manipulation of magnetic particles, combined with the cheap and highly compact lens-free detection makes our system an ideal candidate for point-of-care diagnostic applications.
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ADN/análisis , Magnetismo , Hibridación de Ácido Nucleico/métodos , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
We present a microfluidic platform for the continuous separation of suspended particles based on their size and settling velocity. The separation method takes advantage of the flow field in the vicinity and inside slanted open cavities. These cavities induce flow along them, which deflects the suspended particles to a different degree depending on the extent to which they penetrate into the cavities. The cumulative deflection in the periodic array ultimately leads to vector chromatography, with the different species in the sample moving in different directions. We demonstrate density and size based separation over a range of flow rates by separating polystyrene and silica particles and show that purities nearing 100% can be achieved for multicomponent mixtures. We also demonstrate the potential of the platform to separate biological cells by fractionating different blood components. We discuss the presence of two regimes, depending on the ratio between the settling velocity and the velocity of the particles across the open cavities. The proposed platform could also integrate additional separative force fields in the direction normal to the plane of the cavities to fractionate specific mixtures based on the distinguishing properties of the component species.
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Separación Celular/métodos , Leucocitos/citología , Técnicas Analíticas Microfluídicas/métodos , Separación Celular/instrumentación , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Tamaño de la Partícula , Poliestirenos/química , Dióxido de Silicio/químicaRESUMEN
Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.
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Genotipo , Neoplasias/sangre , Neoplasias/genética , Células Neoplásicas Circulantes , Técnicas Biosensibles , Línea Celular Tumoral , Técnicas Electroquímicas , Humanos , Separación Inmunomagnética/instrumentación , Separación Inmunomagnética/métodos , Neoplasias/diagnóstico , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations.
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ADN Viral/aislamiento & purificación , Magnetismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ADN Viral/genética , Papillomavirus Humano 16/genética , Estreptavidina/químicaRESUMEN
Affinity binding is the principle used in a large number of bio-assays. Aside from specific bindings, non-specific bindings usually deteriorate assays by giving false positive signals and restrict the detection limit. Currently, the assay specificity is mainly dependent on the effectiveness of a suitable surface chemistry. We report an approach to discriminate specific and non-specific bindings with dielectrophoretic (DEP) forces for on-chip magnetic bio-assays. Conjugated to the analytes, magnetic particles were used as the agents for DEP force generation. Due to a weaker binding strength, the non-specifically bound particles were removed while specific bindings remained intact. Analytical and finite element calculations were also performed to study all relevant forces. Furthermore, the removal of magnetic particles was also assessed by measuring the magnetic signal using magnetoresistive sensors. This technique can not only be used to improve the specificity of the on-chip bio-assays but also be developed as a tool of force spectroscopy for the study of bio-molecular binding physics.
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Bioensayo/instrumentación , Diseño Asistido por Computadora , Electroforesis/métodos , Dispositivos Laboratorio en un Chip , Magnetismo/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadAsunto(s)
Separación Celular/instrumentación , Procedimientos Analíticos en Microchip , Células Neoplásicas Circulantes , Biomarcadores de Tumor , Técnicas Biosensibles , Separación Celular/métodos , ADN Complementario/genética , Neoplasias/genética , Neoplasias/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Over the past 5 years, the on-chip detection and manipulation of magnetic beads via magnetoelectronics has emerged as a promising new biosensor platform. Magnetic bead sensing (MBS) provides a highly sensitive and specific technique, enabling these sensors to meet the diagnostic needs that are currently not met by existing technologies. Although many studies have proven the high physical sensitivity of magnetic sensors, the establishment of dose-response curves using MBS is unexplored and their capability to sensitively detect low concentrations of target molecules for diagnostic applications has remained unproven. In this study, we have exploited an alternative MBS concept based on the repositioning of the magnetic beads toward the most sensitive location on the spin valve sensors to allow for highly sensitive immunosensing over a wide range of target concentrations. Furthermore, we present the optimization of the magnetoimmuno assay, i.e., the surface chemistry, the blocking procedure, and the type of magnetic particle, for the highly sensitive and specific detection of S100betabeta, a diagnostic marker for stroke and minor head injury. Finally, a dose-response curve was established that illustrates that our MBS platform can specifically detect S100betabeta down to 27 pg/mL, while maintaining a broad dynamic detection range of approximately 2 decades.