Polybetaine nanobrushes are widely used as inert platforms for label-free biosensing due to their resistance to nonspecific interactions. Despite being considered cationic or electrically neutral, polybetaines can exhibit a negative zeta potential (ZP) at pHs above their isoelectric point (pI). To clarify whether negative zeta potential effectively contributes to surface interactions, we examined three types of nanobrushes deposited on a planar gold substrate: two polybetaines: poly(carboxybetaine methacrylamide) (pCBMAA) and poly(sulfobetaine methacrylamide) (pSBMAA) and hydrophilic poly[N-(2-hydroxypropyl) methacrylamide] (pHPMAA), which carries no ionic group. All three brushes exhibit a well-defined pI and negative surface ZP at pHs above their pI. The pH dependence of the interactions of these brushes with anionic dextran sulfate (DS) and cationic poly[(N-trimethylammonium)ethyl methacrylate] (PTMAEMA) was monitored by infrared reflection spectroscopies (infrared reflection absorption spectroscopy (IRRAS), grazing angle attenuated total reflectance (GAATR)). DS adsorbs to pCBMAA strongly and only weakly to pSBMAA at pHs below their pI but can adsorb slightly to both polybetaines even at pHs above their pI. This is due to the displacement of their carboxylate or sulfo groups from the interaction with the quaternary ammonium cation by the DS sulfate groups. However, DS does not adsorb to pHPMAA at any pH, and PTMAEMA does not adsorb to any of the brushes, regardless of pH. These findings highlight that zeta potential determinations alone may not be sufficient to predict electrostatic interactions as the apparent negative charge does not necessarily translate into a functional surface charge influencing macromolecular interactions.

• Publication type
• Journal Article MeSH

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.

• Keywords
• antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• DNA, Single-Stranded * MeSH
• Humans MeSH
• Surface Plasmon Resonance * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Single-Stranded * MeSH

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Keywords
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• Ixodes * parasitology   MeSH
• Proteome MeSH
• Proteomics MeSH
• Digestive System MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH

BACKGROUND: Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. METHODS: A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. RESULTS: Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4 h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. CONCLUSIONS: Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.

• Keywords
• Prague, SARS-CoV-2, antifouling biosensor, public transportation, qRT-PCR, quartz crystal microbalance, surface contamination,
• MeSH
• COVID-19 * MeSH
• Transportation MeSH
• Humans MeSH
• Pandemics prevention & control   MeSH
• Respiratory Aerosols and Droplets MeSH
• SARS-CoV-2 * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• Keywords
• biosensor, immunoassays, rolling circle amplification, single molecule, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• Biosensing Techniques * methods   MeSH
• DNA, Single-Stranded MeSH
• Limit of Detection MeSH
• Surface Plasmon Resonance methods   MeSH
• Nucleic Acid Amplification Techniques * methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Single-Stranded MeSH