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1.
Cell ; 179(3): 787-799.e17, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31626775

RESUMO

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.


Assuntos
Testes Genéticos , Genômica , NF-kappa B/genética , Fator de Transcrição RelA/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Complexo Mediador/genética , RNA Guia de Cinetoplastídeos/genética
2.
Cell ; 179(6): 1289-1305.e21, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31761534

RESUMO

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.


Assuntos
Adipogenia , Cílios/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Cílios/efeitos dos fármacos , AMP Cíclico/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo
3.
Mol Cancer ; 22(1): 190, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-38017545

RESUMO

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process. METHODS: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens. RESULTS: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01). CONCLUSION: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.


Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Cromatina , Metaloproteinase 8 da Matriz/genética , Neoplasias de Mama Triplo Negativas/genética , Recidiva Local de Neoplasia/genética , Família Multigênica
4.
Nucleic Acids Res ; 43(22): e151, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26240388

RESUMO

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.


Assuntos
Análise Mutacional de DNA/métodos , Oligonucleotídeos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , DNA Mitocondrial/química , DNA de Neoplasias/química , Humanos , Camundongos , Mutação Puntual , RNA Mensageiro/química
5.
Proteomics ; 16(8): 1251-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26935855

RESUMO

Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.


Assuntos
Antígenos/metabolismo , Análise Serial de Proteínas/métodos , Proteoma/metabolismo , Proteômica/métodos , Ligação Competitiva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Magnetismo , Masculino , Microesferas , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes
6.
J Clin Microbiol ; 53(2): 425-32, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25411178

RESUMO

To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.


Assuntos
Antibacterianos/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Meios de Cultura/química , Humanos , Fatores de Tempo
7.
Small ; 10(14): 2877-82, 2014 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-24616417

RESUMO

For the first time DNA coils formed by rolling circle amplification are quantified on-chip by Brownian relaxation measurements on magnetic nanobeads using a magnetoresistive sensor. No external magnetic fields are required besides the magnetic field arising from the current through the sensor, which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chip-based readout is an important step towards the realization of field tests based on rolling circle amplification molecular analyses.


Assuntos
Bacillus/química , DNA Bacteriano/análise , DNA Circular/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Vibrio cholerae/química , Bacillus/genética , Técnicas Biossensoriais/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Circular/química , DNA Circular/genética , Nanopartículas de Magnetita , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico , Esporos Bacterianos/química , Esporos Bacterianos/genética , Vibrio cholerae/genética
8.
BMC Genom Data ; 24(1): 61, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37919672

RESUMO

OBJECTIVES: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited treatment options. Unlike other breast cancer subtypes, the scarcity of specific therapies and greater frequencies of distant metastases contribute to its aggressiveness. We aimed to find epigenetic changes that aid in the understanding of the dissemination process of these cancers. DATA DESCRIPTION: Using CRISPR/Cas9, our experimental approach led us to identify and disrupt an insulator element, IE8, whose activity seemed relevant for cell invasion. The experiments were performed in two well-established TNBC cellular models, the MDA-MB-231 and the MDA-MB-436. To gain insights into the underlying molecular mechanisms of TNBC invasion ability, we generated and characterized high-resolution chromatin interaction (Hi-C) and chromatin accessibility (ATAC-seq) maps in both cell models and complemented these datasets with gene expression profiling (RNA-seq) in MDA-MB-231, the cell line that showed more significant changes in chromatin accessibility. Altogether, our data provide a comprehensive resource for understanding the spatial organization of the genome in TNBC cells, which may contribute to accelerating the discovery of TNBC-specific alterations triggering advances for this devastating disease.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Cromatina/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Mama/metabolismo , Mama/patologia
9.
Nat Biotechnol ; 37(12): 1458-1465, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792411

RESUMO

Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.


Assuntos
Cromatina/genética , Leucemia Aguda Bifenotípica/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Células da Medula Óssea/citologia , Cromatina/química , Análise por Conglomerados , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Epigênese Genética/genética , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genética
10.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570894

RESUMO

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Elementos de Resposta/genética , Linfócitos T/imunologia , Desequilíbrio Alélico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Polissacarídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcriptoma
11.
Nat Commun ; 9(1): 3647, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194434

RESUMO

Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 h on-chip processing time) and library preparation cost (~81¢ per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type.


Assuntos
Montagem e Desmontagem da Cromatina , Ensaios de Triagem em Larga Escala , Imagem Óptica/métodos , Análise de Célula Única/métodos , Animais , Linhagem Celular , Epigênese Genética , Humanos , Camundongos
13.
Methods Mol Biol ; 1616: 147-153, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28600767

RESUMO

Antibiotic susceptibility testing is important to guide clinicians in their choice of antibiotic used for treatment of bacterial infections. Current methods are time-consuming and more rapid alternatives are needed. Here, we describe a novel rapid method for antibiotic susceptibility testing which combines phenotypic and genotypic measurements. The use of padlock probes and rolling circle amplification allows for fast and precise determination of antibiotic susceptibilities as well as species identification.


Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Infecções Urinárias/microbiologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Sondas de DNA , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos
14.
N Biotechnol ; 32(5): 504-10, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25681158

RESUMO

We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.


Assuntos
DNA/análise , Ligadura , Limite de Detecção , Reprodutibilidade dos Testes
15.
Cell Mol Immunol ; 12(4): 493-504, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25958842

RESUMO

Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in nai¨ve mice. Upon immunization with heterologous collagen II in complete Freund's adjuvant the collagen-reactive MZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fcγ receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.


Assuntos
Artrite Reumatoide/imunologia , Subpopulações de Linfócitos B/imunologia , Proliferação de Células , Receptores de IgG/imunologia , Baço/imunologia , Animais , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Subpopulações de Linfócitos B/patologia , Colágeno/toxicidade , Camundongos , Camundongos Mutantes , Receptores de IgG/genética , Baço/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia
16.
ACS Nano ; 9(7): 7374-82, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26166357

RESUMO

We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.


Assuntos
DNA Bacteriano/química , Nanopartículas de Magnetita/química , Técnicas de Diagnóstico Molecular/métodos , Urinálise/métodos , Escherichia coli/genética , Humanos , Fenômenos Ópticos , Proteus mirabilis/genética , Pseudomonas aeruginosa/genética
17.
PLoS One ; 9(11): e111874, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25369034

RESUMO

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , Infecções por Rotavirus/diagnóstico , Rotavirus/genética , Sequência de Bases , Sondas de DNA/genética , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular , Dados de Sequência Molecular , Infecções por Rotavirus/virologia
18.
Biosens Bioelectron ; 29(1): 195-9, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21907556

RESUMO

Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID). It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ∼4pM for all bead sizes under study. The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.


Assuntos
Técnicas Biossensoriais/métodos , DNA Circular/análise , Nanopartículas de Magnetita , DNA Circular/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico , Sondas de Oligonucleotídeos/genética , Tamanho da Partícula
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