The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (μMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the μMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.

• MeSH
• Escherichia coli izolace a purifikace   MeSH
• imunoglobulin G chemie   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• laboratoř na čipu MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• mikrosféry MeSH
• mléko chemie   MeSH
• Salmonella cytologie   imunologie   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

• MeSH
• design vybavení MeSH
• elektrochemické techniky přístrojové vybavení   metody   MeSH
• elektrody MeSH
• imobilizační protilátky chemie   metabolismus   MeSH
• imunoglobulin G chemie   metabolismus   MeSH
• imunoglobuliny chemie   metabolismus   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• králíci MeSH
• kur domácí MeSH
• lidé středního věku MeSH
• lidé MeSH
• metalothionein krev   MeSH
• nádory hlavy a krku krev   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line.

• MeSH
• antigeny nádorové metabolismus   MeSH
• imobilizační protilátky chemie   metabolismus   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• kyseliny polymethakrylové chemie   MeSH
• lidé MeSH
• magnety * MeSH
• mikrofluidní analytické techniky přístrojové vybavení   MeSH
• mikrosféry MeSH
• molekuly buněčné adheze metabolismus   MeSH
• monoklonální protilátky chemie   metabolismus   MeSH
• nádorové cirkulující buňky * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pathogenic bacteria have become a serious socio-economic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn-proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn-proteins by superparamagnetic beads modified with chicken anti-Zn(II) antibody. S. aureus culture was treated with ZnCl(2). Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle-surface interactions were tested and associated protein profiles were identified using SDS-PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS-PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.

• MeSH
• bakteriální proteiny chemie   izolace a purifikace   metabolismus   MeSH
• imunoglobulin G metabolismus   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• kur domácí MeSH
• lidé MeSH
• limita detekce MeSH
• metaloproteiny chemie   izolace a purifikace   metabolismus   MeSH
• protilátky bakteriální metabolismus   MeSH
• robotika přístrojové vybavení   metody   MeSH
• Staphylococcus aureus chemie   izolace a purifikace   metabolismus   MeSH
• zinek chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH