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1.
Anal Chem ; 93(36): 12426-12433, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34470214

RESUMO

The detection of IgG/IgM antibodies is a crucial tool for the diagnosis of infectious diseases as they give specific information such as the stage of infection or when it approximately occurred. In this work, a linear cryogel array (LCA) technology is described for the detection of IgG and IgM antibodies, indicative of a borreliosis infection in human sera. The LCA consists of a transparent capillary filled with functionalized cryogel compartments. For the generation of these cryogel arrays, solutions containing a photo-copolymer and the appropriate antigens are sucked into a surface-modified glass capillary. The solution compartments are separated from each other through air pockets. After freezing the solutions, a photo-induced cross-linking process is performed, through which the solutions are transformed into cryogel compartments, covalently attached to the capillary walls. We show that the LCA technology allows the simultaneous detection of IgG and IgM antibodies via a sandwich immunoassay in sera from Borrelia-infected patients within 1 h for sample sizes of only 12 µL. A study with sera from 42 patients conducted with the LCAs and referenced - depending on the source of the sera - to a commercial line immunoassay and a chemiluminescent immunoassay, which are currently widely used for Lyme disease screening, demonstrates the diagnostic potential of the approach.


Assuntos
Criogéis , Doença de Lyme , Anticorpos Antibacterianos , Humanos , Imunoglobulina M , Doença de Lyme/diagnóstico , Sensibilidade e Especificidade
2.
Langmuir ; 37(37): 11041-11048, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34506153

RESUMO

A platform based on cryogel monoliths in small capillaries, which allows very strong enrichment of an analyte through a capture and release process, is described. For their preparation, a photoreactive copolymer solution containing capture molecules of interest is filled into a capillary, frozen in, and then photochemically transformed into cryogel monoliths through C,H-insertion cross-linking reactions. As a test example, the platform is used for the preconcentration of dopamine from bovine serum albumin and urine samples through capture and release processes. During capture from a large volume and release into a smaller volume, the platform shows recovery rates up to 97% and allows up to a roughly 630-fold enrichment of the concentration of the analyte. The presented platform could be used as a disposable device for the purification and enrichment of a variety of cis-diol-containing samples.


Assuntos
Criogéis , Soroalbumina Bovina
3.
Langmuir ; 37(21): 6510-6520, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34003660

RESUMO

In this study, we present the generation of novel, multifunctional polymer networks through a combination of C,H-insertion cross-linking (CHic) and click chemistry. To this, copolymers consisting of hydrophilic N,N-dimethylacrylamide as matrix component and repeat units containing azide moieties, as well as benzophenone or anthraquinone groups, are generated. The benzophenone or anthraquinone groups allow photo-cross-linking, surface attachment or covalent immobilization of adjacent (bio)molecules through CHic reactions. The azide moieties either can react with available alkynes through conventional click reactions or can be activated to form nitrenes, which can also undergo CHic reactions. By choosing appropriate reaction conditions, the same polymer can be used to follow very different reaction paths, opening up a plethora of choices for the generation of functional polymer networks. In the exemplary presented case ("CHic-Click"), irradiation of the copolymers with UV-A light (λirr = 365 nm) leads to cross-linking (network formation) and surface attachment simultaneously. The azide units remain intact during this cross-linking step, and alkyne-modified (bio)molecules can be bound through click reactions. Biofunctionalization of the polymer network with alkynylated streptavidin, followed by application of biotin-conjugated antibody and a model analyte, highlights the potential of these surface architectures as a toolbox which can be adapted for diverse bioanalytical applications.

4.
Biomacromolecules ; 22(7): 2864-2873, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34126012

RESUMO

We present an alternative to commonly used, but from an environmental point of view, problematic wet strength agents, which are usually added to paper to prevent a loss of mechanical stability and finally disintegrate when they get into contact with water. To this end, diazoester-containing copolymers are generated, which are coated onto paper and by heating to 110-160 °C for short periods of time become activated and form carbene intermediates, which undergo a CH-insertion cross-linking reaction. The process leads to a simultaneous cross-linking of the polymer and its attachment to the cellulose substrate. The immobilization process of copolymers consisting of a hydrophilic matrix based on N,N-dimethylacrylamide and a diazoester-based comonomer to a cellulose model surface and to laboratory-engineered, fibrous paper substrates is investigated as a function of time, temperature, and cross-linker composition. The distribution of the polymer in the fiber network is studied using confocal fluorescence microscopy. Finally, the tensile properties of modified wet and dry eucalyptus sulfate papers are measured to demonstrate the strong effect of the thermally cross-linked copolymers on the wet strength of paper substrates. Initial experiments show that the tensile indices of the modified and wetted paper samples are up to 50 times higher compared to the values measured for unmodified samples. When dry and wet papers coated with the above-described wetting agents are compared, relative wet strengths of over 30% are observed.


Assuntos
Celulose , Água , Interações Hidrofóbicas e Hidrofílicas , Resistência à Tração
5.
Sensors (Basel) ; 21(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34640668

RESUMO

Microfluidic paper combines pump-free water transport at low cost with a high degree of sustainability, as well as good availability of the paper-forming cellulosic material, thus making it an attractive candidate for point-of-care (POC) analytics and diagnostics. Although a number of interesting demonstrators for such paper devices have been reported to date, a number of challenges still exist, which limit a successful transfer into marketable applications. A strong limitation in this respect is the (unspecific) adsorption of protein analytes to the paper fibers during the lateral flow assay. This interaction may significantly reduce the amount of analyte that reaches the detection zone of the microfluidic paper-based analytical device (µPAD), thereby reducing its overall sensitivity. Here, we introduce a novel approach on reducing the nonspecific adsorption of proteins to lab-made paper sheets for the use in µPADs. To this, cotton linter fibers in lab-formed additive-free paper sheets are modified with a surrounding thin hydrogel layer generated from photo-crosslinked, benzophenone functionalized copolymers based on poly-(oligo-ethylene glycol methacrylate) (POEGMA) and poly-dimethyl acrylamide (PDMAA). This, as we show in tests similar to lateral flow assays, significantly reduces unspecific binding of model proteins. Furthermore, by evaporating the transport fluid during the microfluidic run at the end of the paper strip through local heating, model proteins can almost quantitatively be accumulated in that zone. The possibility of complete, almost quantitative protein transport in a µPAD opens up new opportunities to significantly improve the signal-to-noise (S/N) ratio of paper-based lateral flow assays.


Assuntos
Microfluídica , Polímeros , Adsorção , Hidrogéis , Papel
6.
Anal Chem ; 92(15): 10283-10290, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32501674

RESUMO

Droplet-based microfluidic systems offer a high potential for miniaturization and automation. Therefore, they are becoming an increasingly important tool in analytical chemistry, biosciences, and medicine. Heterogeneous assays commonly utilize magnetic beads as a solid phase. However, the sensitivity of state of the art microfluidic systems is limited by the high bead concentrations required for efficient extraction across the water-oil interface. Furthermore, current systems suffer from a lack of technical solutions for sequential measurements of multiple samples, limiting their throughput and capacity for automation. Taking advantage of the different wetting properties of hydrophilic and hydrophobic areas in the channels, we improve the extraction efficiency of magnetic beads from aqueous nanoliter-sized droplets by 2 orders of magnitude to the low µg/mL range. Furthermore, the introduction of a switchable magnetic trap enables repetitive capture and release of magnetic particles for sequential analysis of multiple samples, enhancing the throughput. In comparison to conventional ELISA-based sandwich immunoassays on microtiter plates, our microfluidic setup offers a 25-50-fold reduction of sample and reagent consumption with up to 50 technical replicates per sample. The enhanced sensitivity and throughput of this system open avenues for the development of automated detection of biomolecules at the nanoliter scale.


Assuntos
Automação/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Separação Imunomagnética/métodos , Fenômenos Magnéticos , Técnicas Analíticas Microfluídicas/métodos , Nanoestruturas , Anticorpos/química , Fluorocarbonos/química
7.
Biomacromolecules ; 19(12): 4641-4649, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30433766

RESUMO

In bioanalytical applications, many coating strategies have been established for so-called "blocking" of the surfaces. However, most of the procedures developed so far require additional processing steps for surface blocking and small variations in the blocking efficiency result in increased background noise, which lowers the overall sensitivity of an assay. In this study, we demonstrate the preparation of a bioanalytical surface with a thin film of a photo-cross-linkable copolymer that is transformed photochemically into a surface-attached hydrogel network. The presented coating is directly applicable to various plastic substrates that are used for bioassays without the need for any prior surface modification. Such a strategy allows facile one-step immobilization of biomolecules for bioanalysis and protein-repellent properties for avoiding unspecific adsorption of analyte molecules during the assay. The protein adsorption behavior of the hydrogel-coated and blank surfaces is measured by SPR with human serum and physisorption of labeled detection antibodies. We show that the hydrogel surfaces used lower unspecific background signals and background noise and thus increase the sensitivities of the microarray immunoassays.


Assuntos
Hidrogéis/química , Imunoensaio/métodos , Análise em Microsséries/métodos , Proteínas/imunologia , Adsorção/efeitos dos fármacos , Humanos , Polímeros/química , Proteínas/química , Testes Sorológicos , Especificidade por Substrato , Propriedades de Superfície
8.
Anal Chem ; 89(11): 5697-5701, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28530809

RESUMO

We describe a new microfluidic platform to perform immunochromatographic assays. The platform consists of a linear assembly of small, porous cryogel monoliths functionalized with various biomolecules. The cryogels are anchored in an optically transparent capillary, which serves as the microfluidic carrier. This assembly enables fluid flow by capillary action and simple optical detection. Using an in situ preparation method, individual compartments are generated from small plugs of polymer solutions that are transformed into small individually functionalized cryogel monoliths through a photoinduced cross-linking reaction. In the same reaction step, the monoliths are firmly anchored to the surface of the capillary. As proof-of-concept, a prototype platform is successfully used for the detection of the inflammatory marker interleukin 6 via a sandwich immunoassay. We observe excellent assay performance metrics that include high sensitivity, good linearity, and low variation. We also demonstrate fluid transport solely by passive means, which is a critical attribute for point-of-care diagnostics.


Assuntos
Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Animais , Criogéis , Humanos , Imunoensaio/instrumentação , Interleucina-6/análise , Sistemas Automatizados de Assistência Junto ao Leito
9.
Anal Chem ; 89(3): 1846-1854, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28208267

RESUMO

The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging task. CTCs are, especially at the early stages of cancer development, extremely rare cells hidden in a vast background of regular blood cells. We describe a new strategy for the isolation of CTCs from whole blood. The key component is a medical wire coated with a multilayer assembly that allows highly specific capture of EpCAM (epithelial cell adhesion molecule) positive CTCs from blood. The assembly is generated in a layer-by-layer fashion through photochemically induced C,H insertion reactions and consists of a protective layer, which shields the contacting solution from the metal, a protein resistant layer, which prevents nonspecific interactions with proteins and a layer containing the EpCAM antibodies. In vitro experiments show that these surfaces can capture tumor cells from whole blood with enrichment factors (specifically vs nonspecifically bound cells) of up to about 3000 compared to the number of leucocytes in the blood. The purity of the isolated cells is greater than 90%. After "fishing" them from the blood, the cells, still bound to the wire, can be genetically analyzed. This demonstrates that this strategy might prove useful for next generation sequencing.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias/sangue , Células Neoplásicas Circulantes , Anticorpos/imunologia , Anticorpos Imobilizados/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Materiais Revestidos Biocompatíveis , Ensaio de Imunoadsorção Enzimática , Molécula de Adesão da Célula Epitelial/imunologia , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Humanos , Leucemia/sangue , Leucemia/genética , Leucemia/imunologia , Leucemia/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Ácidos Nucleicos/análise , Reprodutibilidade dos Testes , Propriedades de Superfície
10.
Langmuir ; 30(43): 12804-11, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25300748

RESUMO

Two-phase flow systems are increasingly popular for miniaturized, high-throughput performance of analytical or chemical reactions. In this contribution, we extend a previously described method that allows to increase the range of applications of heterogeneous reactions in two-phase flow, i.e., reactions that rely on isolation and purification of the compound of interest for downstream analysis. Our concept is based on liquid plugs, which serve as miniaturized compartments for the analytical reactions. Purification of the target compound is achieved by extracting the analyte from the aqueous compartments using magnetic beads as solid carriers. In the present paper, we elucidate the influence of parameters such as the polarity of the liquid/liquid and solid/liquid interfaces, the magnetic forces and the fluidic conditions onto the extraction performance. The conditions for reliable extraction and purification of the target compounds are determined. Furthermore, we investigate how to facilitate breaking of the plugs through reduction of the surface tension of the solid/liquid interface. When a lower surface tension is employed, a smaller number of beads is required for the extraction process, which implies a higher sensitivity of the device. In addition, we generate channels with different surface chemistries, which are able to manipulate the flow of the two immiscible liquids. We describe a very simple way to generate such devices and show that we can achieve a transition from segmented flow of plugs to a side-by side flow of the two immiscible liquids, a key requirement for the purification of the compounds.

11.
Int J Biol Macromol ; 259(Pt 1): 128846, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38141714

RESUMO

The growth factor Anterior Gradient 2 (AGR2) has been shown to have an effective role in tissue regeneration, but remained largely unexplored in localized tissue engineering applications. Alginate beads have been proven as safe carriers for protein encapsulation, but they suffer from fragility and uncontrolled protein release. For such alginate systems, little is known about how changes in concentrations and ion-crosslinking affect protein release and accumulation in 3-D matrices. To address these questions, an engineered interpenetrating polymer network (IPN) has been used to synthesize a novel hybrid system consisting of AGR2 loaded beads composed of calcium-crosslinked sodium alginate (SA) and carboxymethyl cellulose (CMC). These beads are embedded in films consisting of SA and polyvinyl alcohol (PVA), using a simple ion gelation technique. We assess protein release kinetics and accumulation within the hybrid system by varying polymer concentrations and cross-linking parameters. The IPN hybrid system maintains controlled release over two weeks, without an initial burst period. Through this approach efficicnt delivery of AGR2 is achieved which in turn effectively mediates cell migration and proliferation, resulting in excellent cell viability and complete wound closure. The described release system opens new perspectives in tissue engineering.


Assuntos
Hidrogéis , Álcool de Polivinil , Preparações de Ação Retardada/farmacologia , Polímeros , Alginatos
12.
Anal Chem ; 85(20): 9469-77, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24083685

RESUMO

In this article, we present a concept which uses liquid plugs as reaction volumes for heterogeneous assay reactions to facilitate time-resolved analysis of biomolecular reactions. For this purpose, the reaction is first compartmentalized to a train of many identical plugs. Therefore, we established a simple fluidic setup build from off-the-shelf available tubing and connectors. It permits reliable formation of plugs and successive dosing of further assay reagents to these compartments (plug volume <5% CV). The time course of the reaction is obtained by routing the plugs successively through a detector. Thereby, the arrival time of a given plug at the detector represents the reaction time of the overall reaction at that moment. Thus, each analyzed plug represents a discrete state of the overall reaction. With this approach, we can achieve a temporal resolution as small as one second, which hardly can be met by conventional analytical methods for analysis of endogenous biological compounds. For analysis of the content of the plugs, we developed a method which allows for heterogeneous assays in two-phase flow. For this purpose, functionalized superparamagnetic beads are enclosed in the plugs for specific binding of the assay product. Purification from supernatant species is achieved by transferring the beads with bound analyte across the phase boundary between aqueous plugs and water-immiscible carrier fluid. We demonstrate this assay principle exemplarily for a sandwich immunoassay (cytokine IL-8). Time-resolved analysis is validated by monitoring a cell-free in vitro expression reaction (turboGFP) in plugs and conventionally in bulk solution. We show that our approach allows for analyzing the entire course of a reaction in a single run. It permits kinetic studies of biological processes with significantly reduced experimental effort and consumption of costly reagents.


Assuntos
Bioensaio/métodos , Nanotecnologia/métodos , Animais , Bovinos , Imunoensaio , Interleucina-8/análise , Fatores de Tempo
13.
ACS Biomater Sci Eng ; 9(11): 6379-6389, 2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37875260

RESUMO

Paper is an ideal candidate for the development of new disposable diagnostic devices because it is a low-cost material, allows transport of the liquid on the device by capillary action, and is environmentally friendly. Today, colorimetric analysis is most often used as a detection method for rapid tests (test strips or lateral flow devices) but usually gives only qualitative results and is limited by a relatively high detection threshold. Here, we describe studies using fluorescence as a readout tool for paper-based diagnostics. We study how the optical readout is affected by light transmission, scattering, and fluorescence as a function of paper characteristics such as thickness (grammage), water content, autofluorescence, and paper type/composition. We show that paper-based fluorescence analysis allows better optical readout compared to that of nitrocellulose, which is currently the material of choice in colorimetric assays. To reduce the loss of analyte molecules (e.g., proteins) due to adsorption to the paper surface, we coat the paper fibers with a protein-repellent hydrogel. For this purpose, we use hydrophilic copolymers consisting of N,N-dimethyl acrylamide and a benzophenone-based cross-linker, which are photochemically transformed into a fiber-attached polymer hydrogel on the paper fiber surfaces in situ. We show that the combination of fluorescence detection and the use of a protein-repellent coating enables sensitive paper-based analysis. Finally, the success of the strategy is demonstrated by using a simple LFD application as an example.


Assuntos
Técnicas Analíticas Microfluídicas , Papel , Técnicas Analíticas Microfluídicas/métodos , Proteínas , Hidrogéis
14.
ACS Appl Mater Interfaces ; 15(48): 55346-55357, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37982803

RESUMO

We present a new platform based on hydrogel beads for multiplex analysis that can be fabricated, barcoded, and functionalized in a single step using a simple microfluidic assembly and a photo-cross-linking process. The beads are generated in a two-phase flow fluidic system and photo-cross-linking of the polymer in the aqueous phase by C,H insertion cross-linking (CHic). The size and shape of the hydrogel particles can be controlled over a wide range by fluidic parameters. During the fabrication of the beads, they are barcoded by using physical and optical barcoding strategies. Magnetic beads and fluorescent particles, which allow identification of the production batch number, are added simultaneously as desired, resulting in complex, multifunctional beads in a one-step reaction. As an example of biofunctionalization, Borrelia antigens were immobilized on the beads. Serum samples that originated from infected and non-infected patients could be clearly distinguished, and the sensitivity was as good as or even better than ELISA, the state of the art in clinical diagnostics. The ease of the one-step production process and the wide range of barcoding parameters offer strong advantages for multiplexed analytics in the life sciences and medical diagnostics.


Assuntos
Hidrogéis , Humanos , Ensaio de Imunoadsorção Enzimática
15.
ACS Appl Mater Interfaces ; 14(22): 25147-25154, 2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35617151

RESUMO

Current developments in precision medicine require the simultaneous detection of an increasing number of biomarkers in heterogeneous, complex solutions, such as blood samples. To meet this need, immunoassays on barcoded hydrogel beads have been proposed, although the encoding and decoding of these barcodes is usually complex and/or resource-intensive. Herein, an efficient method for the fabrication of barcoded, functionalized hydrogel beads is presented. The hydrogel beads are generated using droplet-based microfluidics in combination with photochemically induced C-H insertion reactions, allowing photo-crosslinking, (bio-) functionalization, and barcode integration to be performed in a single step. The generated functionalized beads carry single-color barcodes consisting of green-fluorescent particles of different sizes and concentrations, allowing simple and simultaneous readout with a standard plate reader. As a test example, the performance of barcoded hydrogel beads (3 × 3 matrix) functionalized with capture molecules of interest (e.g., antigens) is investigated for the detection of Lyme-disease-specific antibodies in patient sera. The described barcoding strategy for hydrogel beads does not interfere with the bioanalytical process and captivates by its simplicity and versatility, making it an attractive candidate for multiplex bioanalytical processes.


Assuntos
Hidrogéis , Microfluídica , Anticorpos , Biomarcadores , Humanos , Hidrogéis/química , Imunoensaio/métodos , Microfluídica/métodos
16.
Front Bioeng Biotechnol ; 10: 1045154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36532575

RESUMO

The sensitivity of a PCR based biochip assay relies on the efficiency of PCR amplicons in binding to the microarray spots. The essential factor determining the sensitivity is the amount of single stranded (ss) amplicons available for biochip hybridization. Asymmetric PCR can generate ss-amplicons depending on the ratio of primers used in the amplification process, but this process is often inefficient. We report a novel variant of PCR called the Asymmetric Exponential and Linear Amplification (AELA) which can overcome these issues and generate large amounts of single stranded amplicons. AELA-PCR introduces an amplification strategy that makes use of both exponential and linear amplification of the target nucleic acid. This is done by specifically designed primers and choice of adequate thermal profiles. In conventional PCR with a classical thermal profile, these specifically designed primers will work normally and contribute to an exponential increase of amplicons. A designed sequence extension of one of the primers and a very specific thermal profile, will result in a situation that the extended primer will be the only functional one for amplification, resulting in a linear phase of the amplification process. That is why during this step only one of the two strands of the target is amplified linearly and no longer exponentially. The result of the whole process is an amplification product enriched very strongly in one of the two single strands of the target. These adaptions in PCR are particularly favorable where the generation of ss-DNA/RNA is required. We demonstrate the higher biochip sensitivity of AELA-PCR compared to conventional amplification methods with an example of the Staphylococcus aureus detection on a DNA oligonucleotide microarray.

17.
Langmuir ; 27(10): 6116-23, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21491877

RESUMO

For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.


Assuntos
Proteínas Imobilizadas/química , Polímeros/química , DNA Viral/química , DNA Viral/genética , Histidina-tRNA Ligase/química , Histidina-tRNA Ligase/metabolismo , Humanos , Hidrogéis/química , Proteínas Imobilizadas/metabolismo , Imunoensaio , Iodeto Peroxidase/química , Iodeto Peroxidase/metabolismo , Conformação Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Propriedades de Superfície
18.
Anal Chem ; 82(14): 6124-31, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20552989

RESUMO

Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development.


Assuntos
DNA/análise , Corantes Fluorescentes/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pareamento Incorreto de Bases , Sondas de DNA/química , Reação em Cadeia da Polimerase , Temperatura , Fatores de Tempo
19.
Anal Bioanal Chem ; 397(8): 3533-41, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20596698

RESUMO

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/análise , Replicação de Sequência Autossustentável/métodos , Neoplasias da Mama/genética , Feminino , Células HeLa , Humanos , RNA/genética
20.
Sci Rep ; 9(1): 1246, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718672

RESUMO

Circulating tumor cells (CTCs) play a key role during the metastatic process of human cancers and their reliable detection and characterization could enable new and effective ways of cancer diagnosis, monitoring and treatment. However, due to their ultralow concentration in patient blood, the CTCs must first be enriched before such analysis can be performed. Classical microfiltration is an important and widely used method for the mechanical enrichment of CTCs. This method exploits that CTCs are generally larger than the accompanying blood cells, however, does not differentiate the cells in other ways. In an affinity filtration, selectivity is added by functionalizing the membrane with specific antibodies against a CTC-characteristic surface protein such as the epithelial cell adhesion molecule (EpCAM). A common shortcoming of both filtration approaches is that there is still a poor understanding of the enrichment process and the systems developed so far are frequently operated under non-optimized conditions. To address this, systematic filtration experiments are performed in this work using the EpCAM+ cell line MCF-7 as CTC-model and standard track-etched membranes modified with or without antibodies against EpCAM. The influences of the key filtration parameters time and applied pressure are studied and it is found that in all cases the extent of cell recovery is limited by a lysis process which occurs on the membrane surface. Counterintuitively, it is found that filtration at rather high pressures is advantageous to ensure high recovery rates. To describe the pressure-induced lysis process a biophysical model is developed. This model allows the determination of optimum filtration conditions to achieve both high cancer cell recovery and large blood sample throughput. It is demonstrated that this way practically 100% of spiked cancer cells can be recovered from milliliters of undiluted whole blood within seconds.


Assuntos
Separação Celular/instrumentação , Membranas Artificiais , Células Neoplásicas Circulantes/patologia , Desenho de Equipamento , Filtração/instrumentação , Humanos , Células MCF-7 , Neoplasias/sangue , Neoplasias/patologia
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