Multiplexed electrochemical liposomes applied to the detection of nucleic acids for Influenza A, Influenza B and SARS-CoV-2.

Multiplexing is a relevant strategy for biosensors to improve accuracy and decision-making due to the increased amount of simultaneously obtained information. Liposomes offer unique benefits for label-based multiplexing since a variety of different marker molecules can be encapsulated, leading to intrinsic signal amplification and enabling a variety of detection formats. We successfully developed an electrochemical (EC) liposome-based platform technology for the simultaneous detection of at least three analytes by studying parameters to ensure specific and sensitive bioassay performance. Influenza A and B and SARS-CoV-2 sequences served as model system in a standard sandwich hybridization assay. Studies included encapsulants, probe distribution on liposomes and capture beads, assay setup and interferences between liposomes to also ensure a generalization of the platform. Ruthenium hexamine(III), potassium hexacyanoferrate(II) and m-carboxy luminol, when encapsulated separately into a liposome, provided desirable long-term stability of at least 12 months and no cross-signals between liposomes. Through the optimization process, low limits of detections of 1.6 nmol L-1, 125 pmol L-1 and 130 pmol L-1, respectively, were achieved in a multiplexed assay setup, which were similar to singleplex assays. Non-specific interactions were limited to 25.1%, 7.6% and 7.5%, respectively, through sequential liposome incubations and singleplex capture bead designs. Here, ruthenium hexamine liposomes had only mediocre performance so that low overall signal strength translated into higher LODs and worse specificity. A different marker such as ferroin may be an option in the future. The identification of further electrochemical markers will provide new opportunities for liposomes to function as multiplex, orthogonal or internal standard labels in electrochemical bioassays.

Miniaturized sensor for electroanalytical and electrochemiluminescent detection of pathogens enabled through laser-induced graphene electrodes embedded in microfluidic channels.

High performance, laser-induced graphene (LIG) electrodes were integrated into adhesive tape-based microfluidic channels to realize both electrochemical (EC) and electrochemiluminescent (ECL) detection approaches. This provides strategies for low limits of detection, simple hardware requirements and inexpensive fabrication, which are characteristics required for assays in the competitive point-of-care (POC) sensor field. Here, electrode design and microchannel dimensions were studied and a DNA hybridization assay with liposomes for signal amplification was developed for the specific detection of DNA derived from Cryptosporidium parvum as the model analyte. Liposomes entrapped either Ru(bpy)32+ or K4[Fe(CN)6] generating ECL- and EC-signal amplification, respectively. This new microchip provided all desirable analytical figures of merit needed for POC applications. Specifically, a desirable one-step assay was designed which provided a limit of detection of 3 pmol L-1 for the ECL and 47 pmol L-1 for the EC approach and furthermore enabled highly specific detection considering that at room temperature in this simple setup a single nucleotide polymorphism resulted in a signal decrease of 58%, whereas a decrease of > 98% was observed for non-matching sequences present in 10-fold excess. Direct detection in various matrices ranging from drinking water to soil extracts was also achieved. It is concluded that the simple and inexpensive fabrication in combination with signal amplification strategies makes these concepts relevant for on-site pathogen detection in resource-limited environments.

Shedding Light on the Diversity of Surfactant Interactions with Luminol Electrochemiluminescence for Bioanalysis.

Luminol is a major probe for chemiluminescence (CL) and electrochemiluminescence (ECL) detection technologies in (bio)analysis. Surfactants are added to ECL assay cocktails to enhance signals or are present, owing to given bioassay protocols, yet little is known regarding their effects on luminol ECL. In-depth understanding is provided here through a broad study with bioanalytically relevant surfactants (cationic, anionic, and nonionic), four common electrode materials, and two luminol derivatives. Naturally, in ECL, surface effects are dominant; however, bulk solution, diffusion, and luminescence-stabilization processes also contribute significantly to the overall reaction. It was found that in contrast to CL the effect surfactants have on luminol ECL cannot be linked to general surfactant characteristics such as ionic nature, hydrophilic lipophilic balance (HLB) value, and critical micellar concentration (CMC). Instead, surfactants act in an all-encompassing mechanism, including surface electrochemistry, their solution and interfacial phases, and the chemical luminescence pathway. This leads to dramatic differences in signals obtained, ranging from 5-fold increases to total quenching. Within this complexity, we defined six guiding principles that are extrapolated from the underlying mechanisms and selection guides for surfactant, electrode, and environmental condition combinations. Those will now assist in developing highly sensitive luminol-ECL-based bioassays, because the surfactant selection can be based not only on properties needed for the assay protocol but also on identifying the optimal electrode-surfactant pair to maximize detection efficiency.

Effects of individual glucose levels on the neuronal correlates of emotions.

This study aimed to directly assess the effect of changes in blood glucose levels on the psychological processing of emotionally charged material. We used functional magnetic resonance imaging (fMRI) to evaluate the effect of blood glucose levels on three categories of visually presented emotional stimuli. Seventeen healthy young subjects participated in this study (eight females; nine males; body weight, 69.3 ± 14.9 kg; BMI, 22 ± 2.7; age, 24 ± 3 years), consisting of two functional MRI sessions: (1) after an overnight fast under resting conditions (before glucose administration); (2) after reaching the hyperglycemic state (after glucose administration). During each session, subjects were presented with visual stimuli featuring funny, neutral, and sad content. Single-subject ratings of the stimuli were used to verify the selection of stimuli for each category and were covariates for the fMRI analysis. Analysis of the interaction effect of the two sessions (eu- and hyperglycemia), and the emotional categories accounting for the single-subject glucose differences, revealed a single activation cluster in the hypothalamus. Analysis of the activation profile of the left amygdala corresponded to the three emotional conditions, and this profile was obtained for both sessions regardless of glucose level. Our results indicate that, in a hyperglycemic state, the hypothalamus can no longer respond to emotions. This study offers novel insight for the understanding of disease-related behavior associated with dysregulation of glucose and glucose availability, potentially offering improved diagnostic and novel therapeutic strategies in the future.

Area-specific modulation of neural activation comparing escitalopram and citalopram revealed by pharmaco-fMRI: a randomized cross-over study.

Area-specific and stimulation-dependent changes of human brain activation by selective serotonin reuptake inhibitors (SSRI) are an important issue for improved understanding of treatment mechanisms, given the frequent prescription of these drugs in depression and anxiety disorders. The aim of this neuroimaging study was to investigate differences in BOLD-signal caused by administration of the SSRIs escitalopram and citalopram using pharmacological functional magnetic resonance imaging (pharmaco-fMRI). Eighteen healthy subjects participated in a placebo-controlled, randomized, double-blind study in cross-over repeated measures design. Each volunteer performed facial emotional discrimination and a sensorimotor control paradigm during three scanning sessions. Citalopram (20 mg/d), escitalopram (10 mg/d) and placebo were administered for 10 days each with a drug-free period of at least 21 days. Significant pharmacological effects on BOLD-signal were found in the amygdala, medial frontal gyrus, parahippocampal, fusiform and middle temporal gyri. Post-hoc t-tests revealed decreased BOLD-signal in the right amygdala and left parahippocampal gyrus in both pharmacological conditions, compared to placebo. Escitalopram, compared to citalopram, induced a decrease of BOLD-signal in the medial frontal gyrus and an increase in the right fusiform and left parahippocampal gyri. Drug effects were concentrated in brain regions with dense serotonergic projections. Both escitalopram and citalopram attenuated BOLD-signal in the amygdala and parahippocampal cortex to emotionally significant stimuli compared to control stimuli. We believe that reduced reactivity in the medial frontal gyrus found for escitalopram compared to citalopram administration might explain the response differences between study drugs as demonstrated in previous clinical trials.

Correlations and anticorrelations in resting-state functional connectivity MRI: a quantitative comparison of preprocessing strategies.

Resting-state data sets contain coherent fluctuations unrelated to neural processes originating from residual motion artefacts, respiration and cardiac action. Such confounding effects may introduce correlations and cause an overestimation of functional connectivity strengths. In this study we applied several multidimensional linear regression approaches to remove artificial coherencies and examined the impact of preprocessing on sensitivity and specificity of functional connectivity results in simulated data and resting-state data sets from 40 subjects. Furthermore, we aimed at clarifying possible causes of anticorrelations and test the hypothesis that anticorrelations are introduced via certain preprocessing approaches, with particular focus on the effects of regression against the global signal. Our results show that preprocessing in general greatly increased connection specificity, in particular correction for global signal fluctuations almost doubled connection specificity. However, widespread anticorrelated networks were only found when regression against the global signal was applied. Results in simulated data sets compared with result of human data strongly suggest that anticorrelations are indeed introduced by global signal regression and should therefore be interpreted very carefully. In addition, global signal regression may also reduce the sensitivity for detecting true correlations, i.e. increase the number of false negatives. Concluding from our results we suggest that is highly recommended to apply correction against realignment parameters, white matter and ventricular time courses, as well as the global signal to maximize the specificity of positive resting-state correlations.

Multimodal imaging of human early visual cortex by combining functional and molecular measurements with fMRI and PET.

Receptor distribution patterns of neurotransmitters and distinct functional fields of the human brain appear to be tightly connected with respect to their topological allocation along the cerebral cortex. There is, however, considerable lack of human data directly demonstrating this association in vivo. Here, we assessed the relationship between the distribution of the major inhibitory serotonergic neurotransmitter receptor, the 5-HT(1A) subtype, and the functional organization within early visual cortex defined by retinotopic mapping. The 5-HT(1A) receptor-binding potential was quantified by positron emission tomography (PET) using the highly selective and specific radioligand [carbonyl-(11)C]WAY-100635 in seven healthy subjects. The retinotopic maps and borders determined by functional magnetic resonance imaging (fMRI) were compared to the receptor distribution employing surface-based region of interest analysis in each of these subjects. We found a significant difference in receptor-binding potential in the functionally defined primary (V1) compared to secondary (V2) visual area, as V1 exhibits only 68% of receptor binding found in V2 in both hemispheres, which is consistent with postmortem data. Our in vivo findings clearly support prior assumptions of a link between receptor distribution and functional fields of the human cortex.