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INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times. AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant. EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.
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Proteómica , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Bancos de Muestras Biológicas , Cromatografía Liquida/métodos , Cromatografía Líquida de Alta Presión , Adhesión en Parafina/métodos , Formaldehído/química , Fijación del Tejido/métodosRESUMEN
We present a novel method for the quantitative analysis of mixtures of semivolatile chemical compounds. For the first time, thermal desorption is integrated directly with nanoelectromechanical infrared spectroscopy (NEMS-IR-TD). In this new technique, an analyte mixture is deposited via nebulization on the surface of a NEMS sensor and subsequently desorbed using heating under vacuum. The desorption process is monitored in situ via infrared spectroscopy and thermogravimetric analysis. The resulting spectro-temporal maps allow for selective identification and analysis of the mixture. In addition, the corresponding thermogravimetric data allow for analysis of the desorption dynamics of the mixture components. As a demonstration, caffeine and theobromine were selectively identified and quantified from a mixture with a detection limit of less than 6 pg (about 30 fmol). With its exceptional sensitivity, NEMS-IR-TD allows for the analysis of low abundance and complex analytes with potential applications ranging from environmental sensing to life sciences.
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Espectrofotometría InfrarrojaRESUMEN
The response of triple-negative breast cancer (TNBC) patients to pre-operative (neoadjuvant chemotherapy) is a critical factor of their outcome. To determine the effects of chemotherapy on the tumor genome and to identify mutations associated with chemoresistance and sensitivity, we performed whole exome sequencing on pre/post-chemotherapy tumors and matched lymphocytes from 26 patients. We observed great inter-tumoral heterogeneity with no gene mutated recurrently in more than four tumors besides TP53. Although the degree of response to chemotherapy in residual tumors was associated with more subclonal changes during chemotherapy, there was minimal evolution between pre/post-tumors. Indeed, gene sets enriched for mutations in pre- and post-chemotherapy tumors were very similar and reflected genes involved in the biological process of neurogenesis. Somatically mutated genes present in chemosensitive tumors included COL1A2, PRMD15, APOBEC3B, PALB2 and histone protein encoding genes, while BRCA1, ATR, ARID1A, XRCC3 and genes encoding for tubulin-associated proteins were present in the chemoresistant tumors. We also found that the mutational spectrum of post-chemotherapy tumors was more reflective of matching metastatic tumor biopsies than pre-chemotherapy samples. These findings support a portrait of modest ongoing genomic instability with respect to single-nucleotide variants induced by or selected for by chemotherapy in TNBCs.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Terapia Neoadyuvante , Mutación , Histonas , Inestabilidad Genómica , Citidina Desaminasa , Antígenos de Histocompatibilidad MenorRESUMEN
BACKGROUND: Circulating tumor DNA (ctDNA) offers high sensitivity and specificity in metastatic cancer. However, many ctDNA assays rely on specific mutations in recurrent genes or require the sequencing of tumor tissue, difficult to do in a metastatic disease. The purpose of this study was to define the predictive and prognostic values of the whole-genome sequencing (WGS) of ctDNA in metastatic breast cancer (MBC). METHODS: Plasma from 25 patients with MBC were taken at the baseline, prior to treatment (T0), one week (T1) and two weeks (T2) after treatment initiation and subjected to low-pass WGS. DNA copy number changes were used to calculate a Genomic Instability Number (GIN). A minimum predefined GIN value of 170 indicated detectable ctDNA. GIN values were correlated with the treatment response at three and six months by Response Evaluation Criteria in Solid Tumours assessed by imaging (RECIST) criteria and with overall survival (OS). RESULTS: GIN values were detectable (>170) in 64% of patients at the baseline and were significantly prognostic (41 vs. 18 months OS for nondetectable vs. detectable GIN). Detectable GIN values at T1 and T2 were significantly associated with poor OS. Declines in GIN at T1 and T2 of > 50% compared to the baseline were associated with three-month response and, in the case of T1, with OS. On the other hand, a rise in GIN at T2 was associated with a poor response at three months. CONCLUSIONS: Very early measurements using WGS of cell-free DNA (cfDNA) from the plasma of MBC patients provided a tumor biopsy-free approach to ctDNA measurement that was both predictive of the early tumor response at three months and prognostic.
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Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08-0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075-0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.
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ADN Tumoral Circulante/genética , Neoplasias de la Mama Triple Negativas/genética , Quimioterapia Adyuvante , ADN Tumoral Circulante/sangre , Femenino , Frecuencia de los Genes , Genes p53 , Humanos , Persona de Mediana Edad , Tasa de Mutación , Terapia Neoadyuvante , Pronóstico , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/diagnóstico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Mutación , Medicina de Precisión , Pronóstico , Prueba de Estudio Conceptual , Análisis por Matrices de Proteínas/métodos , Secuenciación Completa del GenomaRESUMEN
PURPOSE: The microenvironment of metastatic breast cancer is incompletely characterized, despite prior evidence that it plays a key role in the biology of metastasis. A major component of the tumor stroma is the carcinoma-associated fibroblast (CAF), which has been shown to communicate with other stromal and cancer cells to create a protumorigenic milieu. Our study was designed to characterize human CAFs from different metastatic sites. EXPERIMENTAL DESIGN: We collected eight carcinoma-associated fibroblasts (mCAFs) from different metastatic sites and compared them with CAFs from primary tumors (pCAFs) and with normal breast fibroblasts (NFs). Molecular profiles and effects on breast cancer cell growth, on response to doxorubicin and on T-cell proliferation were compared. RESULTS: We observed marked differences in mCAFs compared with pCAFs and NFs with respect to in vitro proliferation and effects on breast cancer cell migration, spheroid growth, invasion, response to doxorubicin, and in vivo tumor growth. We found marked transcriptomic differences between mCAFs and pCAFs, including increased expression of IFN-related genes and IGF2 in the former. Cluster analysis revealed two groups of mCAFs, with the liver mCAFs clustering together, with increased PDGFA expression. Treatment with an antibody against insulin-like growth factors (BI836845) inhibited growth of mixed mCAF-tumor cell xenografts in vivo. Also, mCAFs had a suppressive effect on T-cell proliferation. CONCLUSIONS: This is the first comparative analysis of a set of CAFs from metastatic sites in breast cancer. It revealed a marked protumorigenic effect in these mCAFs, which occurs in part through increased expression of IGF2.
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Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/patología , Movimiento Celular , Transición Epitelial-Mesenquimal , Factor II del Crecimiento Similar a la Insulina/metabolismo , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Transcriptoma , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.
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Reprogramación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Perilipina-4/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Reprogramación Celular/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Gotas Lipídicas/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor, and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. To fulfill its potential, greater knowledge about preanalytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted. METHODS: To this end, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (Jewish General Hospital). We evaluated the impact on cfDNA levels of preanalytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from patients with breast cancer. RESULTS: Making use of a novel protocol for cfDNA extraction we increased cfDNA quantities, up to double that of commercial kits. We found that a second centrifugation at 3,000 g on frozen plasma is as efficient as a high-speed (16,000 g) centrifugation on fresh plasma and does not affect cfDNA levels. CONCLUSIONS: These results allow for the implementation of protocols more suitable to the clinical setting. Finally, we found that, unlike targeted gene amplification, whole genome amplification resulted in altered fractional abundance of selected ctDNA variants. IMPACT: Our study of the preanalytical variables affecting cfDNA recovery and testing will significantly enhance the quality and application of ctDNA testing in clinical oncology.
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Recolección de Muestras de Sangre/métodos , ADN Tumoral Circulante/análisis , Neoplasias/sangre , Juego de Reactivos para Diagnóstico/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Recolección de Muestras de Sangre/normas , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/aislamiento & purificación , Humanos , Mutación , Neoplasias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Células Tumorales CultivadasRESUMEN
In many biochip applications, it is advantageous to be able to immobilize biomolecules at specific locations on the surface of solid supports. In this protocol, we describe a photochemical surface patterning procedure based on thiol-ene/yne photochemistry which allows for the simple and rapid selective patterning of biomolecules on thiol-ene solid supports. We describe the preparation of solid supports which are required for the immobilization, including porous monoliths, as well as two different immobilization schemes based on biotin-streptavidin interactions and covalent linkage via free amino groups respectively.
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Dispositivos Laboratorio en un Chip , Análisis por Micromatrices/métodos , Fotoquímica/métodos , Análisis por Micromatrices/instrumentación , Microfluídica/instrumentación , Microfluídica/métodos , Fotoquímica/instrumentación , Polímeros , Compuestos de SulfhidriloRESUMEN
A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.
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Neuropatías Amiloides Familiares/diagnóstico , Técnicas Analíticas Microfluídicas/instrumentación , Prealbúmina/análisis , Humanos , Reproducibilidad de los ResultadosRESUMEN
One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.
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Biomarcadores de Tumor/análisis , Biopsia Guiada por Imagen/métodos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ultrasonografía Intervencional/métodos , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biopsia/métodos , Quimioterapia Adyuvante , Femenino , Humanos , Persona de Mediana Edad , Terapia NeoadyuvanteRESUMEN
To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.
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Pepsina A/metabolismo , Mapeo Peptídico/métodos , Péptidos/análisis , Impresión Tridimensional , Compuestos de Sulfhidrilo/química , Animales , Cromatografía Líquida de Alta Presión , Enzimas Inmovilizadas , Hemoglobinas/metabolismo , Humanos , Dispositivos Laboratorio en un Chip , Espectrometría de Masas , Pepsina A/química , Mapeo Peptídico/instrumentación , Péptidos/metabolismo , Polímeros/químicaRESUMEN
A microfluidic platform combined with synchrotron small-angle X-ray scattering (SAXS) was used for monitoring the continuous production of multilamellar vesicles (MLVs). Their production was fast and started to evolve within less than 0.43 s of contact between the lipids and the aqueous phase. To obtain nanoparticles with a narrow size distribution, it was important to use a modified hydrodynamic flow focusing (HFF) microfluidic device with narrower microchannels than those normally used for SAXS experiments. Monodispersed MLVs as small as 160 nm in size, with a polydispersity index (PDI) of approximately 0.15 were achieved. The nanoparticles produced were smaller and had a narrower size distribution than those obtained via conventional bulk mixing methods. This microfluidic platform therefore has a great potential for the continuous production of monodispersed NPs.
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The increasingly narrow and brilliant beams at X-ray facilities reduce the requirements for both sample volume and data acquisition time. This creates new possibilities for the types and number of sample conditions that can be examined but simultaneously increases the demands in terms of sample preparation. Microfluidic-based sample preparation techniques have emerged as elegant alternatives that can be integrated directly into the experimental X-ray setup remedying several shortcomings of more traditional methods. We review the use of microfluidic devices in conjunction with X-ray measurements at synchrotron facilities in the context of 1) mapping large parameter spaces, 2) performing time resolved studies of mixing-induced kinetics, and 3) manipulating/processing samples in ways which are more demanding or not accessible on the macroscale. The review covers the past 15 years and focuses on applications where synchrotron data collection is performed in situ, i.e. directly on the microfluidic platform or on a sample jet from the microfluidic device. Considerations such as the choice of materials and microfluidic designs are addressed. The combination of microfluidic devices and measurements at large scale X-ray facilities is still emerging and far from mature, but it definitely offers an exciting array of new possibilities.
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Disciplinas de las Ciencias Biológicas/instrumentación , Dispositivos Laboratorio en un Chip , Cristalografía por Rayos X , Dispersión del Ángulo Pequeño , Rayos XRESUMEN
The marriage of highly sensitive biosensor designs with the versatility in sample handling and fluidic manipulation offered by lab-on-a-chip systems promises to yield powerful tools for analytical and, in particular, diagnostic applications. The field where these two technologies meet is rapidly and almost violently developing. Yet, solutions where the full potentials are being exploited are still surprisingly rare. In the context of this review, sensor designs are often fairly advanced, whereas the lab-on-a-chip aspect is still rather simplistic in many cases, albeit already offering significant improvements to existing methods. Recent examples, showing a staggering variety of lab-on-a-chip systems for biosensing applications, are presented, tabularized for overview, and briefly discussed.
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Técnicas Biosensibles , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
A novel, rapid and simple method for the preparation of emulsion-templated monoliths in microfluidic channels based on thiol-ene chemistry is presented. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single photoinitiated step. Characterization by scanning electron microscopy showed that the methanol-based emulsion templating process resulted in a network of highly interconnected and regular thiol-ene beads anchored solidly inside thiol-ene microchannels. Surface area measurements indicate that the monoliths are macroporous, with no or little micro- or mesopores. As a demonstration, galactose oxidase and peptide-N-glycosidase F (PNGase F) were immobilized at the surface of the synthesized thiol-ene monoliths via two different mechanisms. First, cysteine groups on the protein surface were used for reversible covalent linkage to free thiol functional groups on the monoliths. Second, covalent linkage was achieved via free primary amino groups on the protein surface by means of thiol-ene click chemistry and l-ascorbic acid linkage. Thus prepared galactose oxidase and PNGase F microreactors demonstrated good enzymatic activity in a galactose assay and the deglycosilation of ribonuclease B, respectively.
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Ascomicetos/enzimología , Proteínas Fúngicas/química , Galactosa/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Ribonucleasas/química , Emulsiones/químicaAsunto(s)
Investigación Biomédica , Biopsia , Consentimiento Informado , Recuerdo Mental , Adulto , Anciano , Anciano de 80 o más Años , Investigación Biomédica/ética , Neoplasias de la Mama/diagnóstico , Femenino , Humanos , Consentimiento Informado/ética , Persona de Mediana Edad , Proyectos Piloto , Investigación CualitativaRESUMEN
Thiol-ene polymers possess physical, optical, and chemical characteristics that make them ideal substrates for the fabrication of optofluidic devices. In this work, thiol-ene polymers are used to simultaneously create microfluidic channels and optical waveguides in one simple moulding step. The reactive functional groups present at the surface of the thiol-ene polymer are subsequently used for the rapid, one step, site-specific functionalization of the waveguide with biological recognition molecules. It was found that while the bulk properties and chemical surface properties of thiol-ene materials vary considerably with variations in stoichiometric composition, their optical properties remain mostly unchanged with an average refractive index value of 1.566 ± 0.008 for thiol-ene substrates encompassing a range from 150% excess ene to 90% excess thiol. Microfluidic chips featuring thiol-ene waveguides were fabricated from 40% excess thiol thiol-ene to ensure the presence of thiol functional groups at the surface of the waveguide. Biotin alkyne was photografted at specific locations using a photomask, directly at the interface between the microfluidic channel and the thiol-ene waveguide prior to conjugation with fluorescently labeled streptavidin. Fluorescence excitation was achieved by launching light through the thiol-ene waveguide, revealing bright fluorescent patterns along the channel/waveguide interface.
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Técnicas Biosensibles/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Polímeros/química , Espectrometría de Fluorescencia/instrumentación , Compuestos de Sulfhidrilo/química , Refractometría , Propiedades de SuperficieRESUMEN
The suitable optical properties of thiol-ene polymers combined with the ease of modifying their surface for the attachment of recognition molecules make them ideal candidates in many biochip applications. This paper reports the rapid one-step photochemical surface patterning of biomolecules in microfluidic thiol-ene chips. This work focuses on thiol-ene substrates featuring an excess of thiol groups at their surface. The thiol-ene stoichiometric composition can be varied to precisely control the number of surface thiol groups available for surface modification up to an average surface density of 136 ± 17 SH nm(-2). Biotin alkyne was patterned directly inside thiol-ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent wave-induced fluorescence (EWIF), demonstrating the success of the grafting procedure and its potential for biochip applications.