biointerface
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The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.
- Klíčová slova
- antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
- MeSH
- jednovláknová DNA * MeSH
- lidé MeSH
- povrchová plasmonová rezonance * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- jednovláknová DNA * MeSH
New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.
- Klíčová slova
- SARS-CoV-2, functional coatings, piezoelectric biosensor, polymer brush, rapid detection, zwitterionic materials,
- MeSH
- biosenzitivní techniky MeSH
- biotest MeSH
- bioznečištění MeSH
- COVID-19 diagnóza MeSH
- fosfoproteiny chemie MeSH
- hmotnostní spektrometrie MeSH
- ionty MeSH
- koronavirové nukleokapsidové proteiny chemie MeSH
- lidé MeSH
- limita detekce MeSH
- nazofarynx virologie MeSH
- nosní sliznice virologie MeSH
- odběr biologického vzorku MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- polymery chemie MeSH
- reprodukovatelnost výsledků MeSH
- RNA virová metabolismus MeSH
- SARS-CoV-2 * MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfoproteiny MeSH
- ionty MeSH
- koronavirové nukleokapsidové proteiny MeSH
- nucleocapsid phosphoprotein, SARS-CoV-2 MeSH Prohlížeč
- polymery MeSH
- RNA virová MeSH
The use of polymers in all aspects of daily life is increasing considerably, so there is high demand for polymers with specific properties. Polymers with antibacterial properties are highly needed in the food and medical industries. Low-density polyethylene (LDPE) is widely used in various industries, especially in food packaging, because it has suitable mechanical and safety properties. Nevertheless, the hydrophobicity of its surface makes it vulnerable to microbial attack and culturing. To enhance antimicrobial activity, a progressive surface modification of LDPE using the antimicrobial agent grafting process was applied. LDPE was first exposed to nonthermal radio-frequency (RF) plasma treatment to activate its surface. This led to the creation of reactive species on the LDPE surface, resulting in the ability to graft antibacterial agents, such as ascorbic acid (ASA), commonly known as vitamin C. ASA is a well-known antioxidant that is used as a food preservative, is essential to biological systems, and is found to be reactive against a number of microorganisms and bacteria. The antimicrobial effect of grafted LDPE with ASA was tested against two strong kinds of bacteria, namely, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), with positive results. Surface analyses were performed thoroughly using contact angle measurements and peel tests to measure the wettability or surface free energy and adhesion properties after each modification step. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to analyze the surface morphology or topography changes of LDPE caused by plasma treatment and ASA grafting. Surface chemistry was studied by measuring the functional groups and elements introduced to the surface after plasma treatment and ASA grafting, using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). These results showed wettability, adhesion, and roughness changes in the LDPE surface after plasma treatment, as well as after ASA grafting. This is a positive indicator of the ability of ASA to be grafted onto polymeric materials using plasma pretreatment, resulting in enhanced antibacterial activity.
- Klíčová slova
- antibacterial, biointerface, grafting modification, plasma treatment, polyethylene,
- Publikační typ
- časopisecké články MeSH
Biocompatibility tests and a study of the electrical properties of thin films prepared from six electroactive polymer ink formulations based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) were performed. The aim was to find a suitable formulation of PEDOT:PSS and conditions for preparing thin films in order to construct printed bioelectronic devices for biomedical applications. The stability and electrical properties of such films were tested on organic electrochemical transistor (OECT)-based sensor platforms and their biocompatibility was evaluated in assays with 3T3 fibroblasts and murine cardiomyocytes. It was found that the thin films prepared from inks without an additive or any thin film post-treatment provide limited conductivity and stability for use in biomedical applications. These properties were greatly improved by using ethylene glycol and thermal annealing. Addition or post-treatment by ethylene glycol in combination with thermal annealing provided thin films with electrical resistance and a stability sufficient to be used in sensing of animal cell physiology. These films coated with collagen IV showed good biocompatibility in the assay with 3T3 fibroblasts when compared to standard cell culture plastics. Selected films were then used in assays with murine cardiomyocytes. We observed that these cells were able to attach to the PEDOT:PSS films and form an active sensor element. Spontaneously beating clusters were formed, indicating a good physiological status for the cardiomyocyte cells. These results open the door to construction of cheap printed electronic devices for biointerfacing in biomedical applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1121-1128, 2018.
- Klíčová slova
- biocompatibility, biointerface, biomedical applications, biosensor, conducting polymer,
- MeSH
- bicyklické sloučeniny heterocyklické chemie MeSH
- biokompatibilní materiály farmakologie MeSH
- buněčné linie MeSH
- buňky 3T3 MeSH
- elektrická impedance MeSH
- elektřina * MeSH
- inkoust * MeSH
- myši MeSH
- polymery chemie MeSH
- polystyreny chemie MeSH
- testování materiálů * MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bicyklické sloučeniny heterocyklické MeSH
- biokompatibilní materiály MeSH
- poly(3,4-ethylene dioxythiophene) MeSH Prohlížeč
- polymery MeSH
- polystyrene sulfonic acid MeSH Prohlížeč
- polystyreny MeSH
- voda MeSH
Nanostructured TiO2 nanotubes (NTs) of diameters from 15 to 100nm were fabricated by an electrochemical anodization process. Biofilm-positive strains of Bacillus cereus and Pseudomonas aeruginosa behaved similarly on all TiO2 NTs as well as on native titanium (Ti) foil. The adhesion and growth of mesenchymal stem cells (MSc), embryonic stem cells (ESc), and pure cardiomyocytes derived from ESc exhibited significant differences. MSc as well as ESc were, in contrast to cardiomyocytes, able to adhere, and grow on TiO2 NTs. A correlation between NTs diameter and cell behaviour was however observed in the case of MSc and ESc. MSc were not in a physiological state in the case of 100nm TiO2 NTs, while ESc were not able to grow on 15nm TiO2 NTs. It can be stated that these differences can be assigned to different diameters of the NTs but not to the chemistry of the surface. This is the first study describing the comprehensive behaviour of both eukaryotic and prokaryotic cells on TiO2 NTs. On the basis of obtained results, it can be concluded that new generation of medical devices providing selective cell behaviour can be fabricated by optimizing the nanoscale morphology of TiO2.
- Klíčová slova
- Biofilm, Nanotubes, Stem cells, Surface morphology, Titanium,
- MeSH
- Bacteria MeSH
- biofilmy MeSH
- kmenové buňky MeSH
- lidé MeSH
- nanostruktury * MeSH
- nanotrubičky MeSH
- titan MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- titan MeSH
- titanium dioxide MeSH Prohlížeč
A plasmonic biosensor for rapid detection of protein biomarkers in complex media is reported. Clinical serum samples were analyzed by using a novel biointerface architecture based on poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] brushes functionalized with bioreceptors. This biointerface provided an excellent resistance to fouling even after the functionalization and allowed for the first time the direct detection of antibodies against hepatitis B surface antigen (anti-HBs) in clinical serum samples using surface plasmon resonance (SPR). The fabricated SPR biosensor allowed discrimination of anti-HBs positive and negative clinical samples in 10min. Results are validated by enzyme-linked immunoassays of the sera in a certified laboratory. The sensor could be regenerated by simple treatment with glycine buffer.
- Klíčová slova
- Antifouling, Biosensors, Blood serum, Hepatitis B, Polymer brushes, Surface plasmon resonance,
- MeSH
- akrylamidy chemie MeSH
- design vybavení MeSH
- hepatitida B - antigeny povrchové imunologie MeSH
- hepatitida B krev imunologie MeSH
- lidé MeSH
- limita detekce MeSH
- povrchová plasmonová rezonance přístrojové vybavení MeSH
- povrchové vlastnosti MeSH
- protilátky virové krev imunologie MeSH
- virus hepatitidy B imunologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
- Názvy látek
- akrylamidy MeSH
- hepatitida B - antigeny povrchové MeSH
- methacrylamide MeSH Prohlížeč
- protilátky virové MeSH
Recent progress in biointerface research has highlighted the role of antifouling functionalizable coatings in the development of advanced biosensors for point-of-care bioanalytical and biomedical applications dealing with real-world complex samples. The resistance to nonspecific adsorption promotes the biorecognition performance and overall increases the reliability and specificity of the analysis. However, the process of modification with biorecognition elements (so-called functionalization) may influence the resulting antifouling properties. The extent of these effects concerning both functionalization procedures potentially changing the surface architecture and properties, and the physicochemical properties of anchored biorecognition elements, remains unclear and has not been summarized in the literature yet. This critical review summarizes these key functionalization aspects with respect to diverse antifouling architectures showing low or ultra-low fouling quantitative characteristics in complex biological media such as bodily fluids or raw food samples. The subsequent discussion focuses on the impact of functionalization on fouling resistance. Furthermore, this review discusses some of the drawbacks of available surface sensitive characterization methods and highlights the importance of suitable assessment of the resistance to fouling.
Poly(1,2,3-triazole)-based polymers remain underexplored as a versatile platform for biomedical soft materials. Here, we report the modular synthesis of amphiphilic poly(1,2,3-triazole)-co-polytriazolium copolymers (qH-PETH) via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) polyaddition and post-polymerization quaternization with a degree of quaternization equal to (0.85). These polymers exhibit unusual visible colouration in the absence of chromophores-appearing almost colourless in N-methyl-2-pyrrolidone (NMP), orange in DMSO, and pale orange in water-accompanied by a pronounced red shift in non-polar solvents such as dichloromethane (DCM) or chloroform. This optical behaviour is attributed to solvent-dependent supramolecular aggregation. Dynamic light scattering (DLS) measurements reveal hydrodynamic diameters of <20 nm in water and approximately 500 nm in chloroform for quaternized polymers (qH-PETH), indicating strong aggregation in low-polarity environments. UV-vis spectroscopy and atomic force microscopy (AFM) further support aggregation-induced optical effects modulated by solvent polarity and hydrogen-bonding capacity. Both neutral and quaternized polymers display excellent cytocompatibility toward human cell lines (HeLa, HEK293, THP-1) at concentrations up to 100 μg mL-1, with no detectable immunogenic activation. These findings position poly(1,2,3-triazole)-co-polytriazolium copolymers as a promising, tunable class of biocompatible soft materials for biointerface engineering and responsive biomedical applications.
- Publikační typ
- časopisecké články MeSH
We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.
- Klíčová slova
- biosensor, immunoassays, rolling circle amplification, single molecule, surface plasmon resonance, surface plasmon-enhanced fluorescence,
- MeSH
- biosenzitivní techniky * metody MeSH
- jednovláknová DNA MeSH
- limita detekce MeSH
- povrchová plasmonová rezonance metody MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- jednovláknová DNA MeSH
