Correction: 'Semen rheology and its relation to male infertility' (2022), by Tomaiuolo et al.

[This corrects the article DOI: 10.1098/rsfs.2022.0048.][This corrects the article DOI: 10.1098/rsfs.2022.0048.].

Organic Electrochemical Transistor Immuno-Sensors for Spike Protein Early Detection.

The global COVID-19 pandemic has had severe consequences from the social and economic perspectives, compelling the scientific community to focus on the development of effective diagnostics that can combine a fast response and accurate sensitivity/specificity performance. Presently available commercial antigen-detecting rapid diagnostic tests (Ag-RDTs) are very fast, but still face significant criticisms, mainly related to their inability to amplify the protein signal. This translates to a limited sensitive outcome and, hence, a reduced ability to hamper the spread of SARS-CoV-2 infection. To answer the urgent need for novel platforms for the early, specific and highly sensitive detection of the virus, this paper deals with the use of organic electrochemical transistors (OECTs) as very efficient ion-electron converters and amplifiers for the detection of spike proteins and their femtomolar concentration. The electrical response of the investigated OECTs was carefully analyzed, and the changes in the parameters associated with the transconductance (i.e., the slope of the transfer curves) in the gate voltage range between 0 and 0.3 V were found to be more clearly correlated with the spike protein concentration. Moreover, the functionalization of OECT-based biosensors with anti-spike and anti-nucleocapside proteins, the major proteins involved in the disease, demonstrated the specificity of these devices, whose potentialities should also be considered in light of the recent upsurge of the so-called "long COVID" syndrome.

Correction: Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid.

Correction for 'Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid' by Giovanna Tomaiuolo et al., Soft Matter, 2023, https://doi.org/10.1039/d3sm00443k.

Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid.

The rheological behavior of human semen is overlooked and essentially unexplored in the scientific literature. Here, we provide the first quantitative experimental evidence that post-liquafaction normospermic human semen behaves as a viscoelastic fluid and the shear moduli can be scaled according to the weak-gel model.

Immuno-Sensing at Ultra-Low Concentration of TG2 Protein by Organic Electrochemical Transistors.

Transglutaminase 2 (TG2) is a ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity. Its subcellular localization is crucial in determining its function, and indeed, TG2 is found in the extracellular matrix, mitochondria, recycling endosomes, plasma membrane, cytosol, and nucleus because it is associated with cell growth, differentiation, and apoptosis. It is involved in several pathologies, such as celiac disease, cardiovascular, hepatic, renal, and fibrosis diseases, carrying out opposite functions of up and down regulation in the progression of the same pathology. Therefore, this fine regulation requires a very sensitive and specific method of identification of TG2, which is to be detected in very small quantities in a deregulated condition. Here, we demonstrate the possibility of detecting TG2 down to attomolar concentration by using organic electrochemical transistors driven by gold electrodes functionalized with anti-TG2 antibodies. In particular, a direct correlation between the TG2 concentration and the transistor transconductance values, as extracted from typical transfer curves, was found. Overall, our findings highlight the potentialities of this new biosensing approach for the detection of TG2 in the context of pathological diseases, offering a rapid and cost-effective alternative to traditional methods.

Effect of surfactant concentration on diffusion and microstructure in water-in-oil emulsions studied by low-field benchtop NMR and optical microscopy.

Emulsions are ubiquitous in many consumer products, including food, cosmetics and pharmaceuticals. Whilst their macroscopic characterisation is well-established, understanding their microscopic behaviour is very challenging. In our previous work we investigated oil-in-water emulsions by studying the effect of water on structuring and dynamics of such systems. In the present work, we investigate the effect of surfactant concentration on microstructure and diffusion within the water-in-oil emulsion system by using low-field pulsed-field gradient (PFG) NMR studies carried out with a benchtop NMR instrument, in conjunction with optical imaging. The results reveal that at high surfactant concentration the formation of smaller droplets gives rise to a third component in the PFG NMR attenuation plot, which is mostly attributed to restricted diffusion near the droplet boundaries. In addition, structuring effects due to increase in surfactant concentration at the boundaries could also contribute to further slowing down water diffusion at the boundaries. As the surfactant concentration decreases, the average droplet size becomes larger and both restriction and structuring effects at the droplet boundaries become less significant, as suggested by the PFG NMR plot, whereby the presence of a third diffusion component becomes less pronounced.

Semen rheology and its relation to male infertility.

Infertility affects 15% of couples of reproductive age worldwide. In spite of many advances in understanding and treating male infertility, there is still a number of issues that need further investigation and translation to the clinic. Here, we review the current knowledge and practice concerning semen rheology and its relation with pathological states affecting male infertility. Although it is well recognized that altered rheological properties of semen can impair normal sperm movement in the female reproductive tract, routine semen analysis is mostly focused on number, motility and morphology of spermatozoa, and includes only an approximate, operator-dependent measure of semen viscosity. The latter is based on the possible formation of a liquid thread from a pipette where a semen sample has been aspirated, a method that is sensitive not only to viscosity but also to elongational properties and surface tension of semen. The formation of a liquid thread is usually associated with a gel-like consistency of the sample and changes in spermatozoa motility in such a complex medium are still to be fully elucidated. The aim of this review is to point out that a more quantitative and reliable characterization of semen rheology is in order to improve the current methods of semen analysis and to develop additional tools for the diagnosis and treatment of male infertility.

Organic electrochemical transistors as novel biosensing platforms to study the electrical response of whole blood and plasma.

In this paper, for the first time to the best of our knowledge, organic electrochemical transistors are employed to investigate the electrical response of human blood, plasma and alternative buffer solutions that inhibit red blood cell (RBC) aggregation. Our focus is on selecting a suitable electrolytic platform and the related operating conditions, where the RBC effect on the OECT response can be observed separately from the strong ionic environment of plasma in whole blood. The transient response of whole blood to pulse experiments is characterized by two time constants, which can be related to blood viscosity and to the capacitive coupling between the ionic and electronic components of the overall system. The role of capacitive effects, likely due to enhanced double-layer formation by negatively charged RBCs, is also confirmed by the increase of transconductance which was found in RBC suspensions as compared to the suspending buffer. Overall, the complex behavior found in these experiments provides new insights for the development of innovative blood-based sensing devices for biomedical applications.

The microstructure of Carbopol in water under static and flow conditions and its effect on the yield stress.

In this work, experimental observations of the microstructure of neutralized polyacrylic acid (Carbopol) in water by confocal microscopy under both static and flow conditions are presented. In the former case, a Carbopol-rich phase made by swollen particles dispersed in a water-rich continuous phase is found, so that the system will be henceforth referred to as a suspension, as long as particles are observed. The swollen particles form dendritic-like aggregates, which span the entire solution volume above a critical concentration. In such conditions, a percolated network can be formed, leading to the onset of a yield stress behavior. By separating the dispersed and continuous phase through centrifugation, we provide evidence of a miscibility gap in the phase behavior of Carbopol in water. When the Carbopol suspensions flow in a microfluidic capillary, a particle-concentrated plug core can be distinguished from a less concentrated layer corresponding to a steep velocity decrease. Confocal imaging also shows that the apparent slip found in Carbopol suspensions is due to a particle-concentrated near-wall region, where no flow is observed. Such flow-induced microstructure is responsible for the different nature of the yield stress values measured by classical rheometry and by flow velocimetry. While the yield stress measured by the former can be here related to the presence of a percolated network, the yield stress obtained from the velocity profile is due to the heterogeneous particle distribution along the capillary radius. These results provide a novel insight on the mechanisms governing yield stress in complex fluids.

Microstructure evolution during nano-emulsification by NMR and microscopy.

HYPOTHESIS: Microstructure evolution in emulsions as a function of composition is of great interest but fundamentals have not yet been fully elucidated. Here, pulsed-field gradient (PFG) NMR diffusion measurements have been combined with confocal laser scanning microscopy (CLSM) to assess evolution of dynamics and microstructure during nano-emulsification. EXPERIMENTS: Diffusion coefficients of emulsions made of water, mineral oil and surfactants (Span 20 and Tween 80) were measured as a function of water composition and compared with the morphological features of the emulsions obtained by CLSM. FINDINGS: In the absence of water, two phases are visible from CLSM, and two diffusion components are observed with PFG NMR, a major fast component attributed to a continuous oil phase containing the more hydrophobic surfactant Span 20 with traces of Tween 80, and a minor slow component attributed to a dispersed phase of the more hydrophilic surfactant Tween 80 with traces of mineral oil and Span 20. At the inversion point (25 wt% water) the two-component diffusion behavior of the oil-rich phase is drastically reversed in terms of populations, with the slow diffusion process becoming dominant. This suggests a significant structuring of the oil-rich phase in the presence of surfactants enhanced by water, which can be explained by the formation of aggregates in the oil phase as reverse micelles or of a lamellar structure, and ties in well with the rheological measurements.

Confined flow behaviour of droplets in microcapillary flow.

The problem of droplets flowing in a micritions is relevant in several applications including flow in porous media. When the flow in the capillary is laminar with negligible gravity effects, droplet velocity and deformation depend upon three independent parameters: the droplet size relative to the capillary radius [Formula: see text][Formula: see text], which is a measure of confinement, the viscosity ratio [Formula: see text] between the droplet and the continuous phase and the capillary number Ca which measures the ratio of viscous to capillary forces. Although droplet microconfined flow behaviour in capillaries has been widely investigated by theoretical models, experimental results are still scarce. Here, an experimental campaign focused on the flow behaviour of axisymmetric confined droplets flowing in a microcapillary is carried out. Our experimental results were obtained by using a water in soybean oil emulsion with a low viscosity ratio and the effect of the aforementioned three parameters, [Formula: see text], [Formula: see text] and Ca, on droplet motion was investigated. Moreover, our experimental results are compared with numerical solutions available in the literature.

Measuring Interfacial Tension of Emulsions in Situ by Microfluidics.

Interfacial tension is a key parameter affecting industrially relevant properties of emulsions, such as morphology and stability. Although several methods are available to measure interfacial tension, they are based on generation of droplets starting from separate emulsion components and cannot directly probe the interfacial tension of an emulsion as such. Here, a novel microfluidic tensiometry device to measure interfacial tension of a water-in-oil emulsion in situ as a function of surfactant concentration is presented. In our approach, interfacial tension is obtained from a quantitative analysis of the deformation of individual emulsion droplets under steady state shear flow in microfluidic channels. The technique is validated by comparing the results with experimental data obtained by the pendant drop method in a broad range of interfacial tension values. A very good agreement is found, and an estimate of the surfactant critical micellar concentration (CMC) is also obtained. The proposed microfluidic setup can be used even at high surfactant concentrations, where the measurement is made more challenging by sample viscoelasticity, thus providing a powerful tool to determine the interfacial tension of complex systems in an extended concentration range. The technique could be also used for in-line monitoring of emulsion processing.

Emulsions in porous media: From single droplet behavior to applications for oil recovery.

Emulsions are suspensions of droplets ubiquitous in oil recovery from underground reservoirs. Oil is typically trapped in geological porous media where emulsions are either formed in situ or injected to elicit oil mobilization and thus enhance the amount of oil recovered. Here, we briefly review basic concepts on geometrical and wetting features of porous media, including thin film stability and fluids penetration modes, which are more relevant for oil recovery and oil-contaminated aquifers. Then, we focus on the description of emulsion flow in porous media spanning from the behaviour of single droplets to the collective flow of a suspension of droplets, including the effect of bulk and interfacial rheology, hydrodynamic and physico-chemical interactions. Finally, we describe the particular case of emulsions used in underground porous media for enhanced oil recovery, thereby discussing some perspectives of future work. Although focused on oil recovery related topics, most of the insights we provide are useful towards remediation of oil-contaminated aquifers and for a basic understanding of emulsion flow in any kind of porous media, such as biological tissues.

The effect of flow on viscoelastic emulsion microstructure.

Emulsions made of oil, water and surfactants are widespread soft materials with complex structures depending on composition and temperature. Emulsion phase behavior at rest has been widely investigated but flow-induced effects, which are very relevant in many applications, can still be further explored towards improved emulsion microstructural design. In this work, we use low energy emulsification processing to create small-sized emulsions. In a previous report, we showed the emulsion morphology development and the effect of flow on the microstructure of a highly viscoelastic attractive emulsion which result in a concentrated nanoemulsion after viscoelastic droplet filaments are disrupted. Here, we show that upon stopping the flow, the filaments slowly buckle, recoil and finally form clusters of randomly flocculated droplets. We thus obtain two completely different emulsion morphologies simply induced by the action of flow, where in both cases attractive interactions play a key role. The emulsion high interfacial area represents a valuable feature for several applications such as upstream operations, microreaction media and drug delivery.

Flow-induced nanostructuring of gelled emulsions.

Although the phase behavior of emulsions has been thoroughly investigated, the effect of flow on emulsion morphology, which is relevant for many applications, is far from being fully elucidated. Here, we investigate an emulsion based on two common nonionic surfactants in a range of water concentration where complex and diverse microstructures are found at rest, such as multilamellar and bicontinuous phases. In spite of such complexity, once subjected to shear flow, all the emulsions investigated are characterized by thinning filaments which eventually break up into a concentrated suspension of micro-sized water-based droplets dispersed in a continuous oil phase. The so-formed droplets tend to align in string-like structures. The emulsions exhibit a yield stress, whose value can be estimated by the plug-core velocity profiles in pressure-driven capillary flow, thus providing evidence of weakly attractive interdroplet interactions. The latter are consistent with droplet clustering and percolation observed at rest. These results can also be relevant to the flow behavior of other liquid-liquid systems, such as polymer blends, where the flow-induced microstructure is under debate as well.

Using optical tweezers for the characterization of polyelectrolyte solutions with very low viscoelasticity.

Recently, optical tweezing has been used to provide a method for microrheology addressed to measure the rheological properties of small volumes of samples. In this work, we corroborate this emerging field of microrheology by using these optical methods for the characterization of polyelectrolyte solutions with very low viscoelasticity. The influence of polyelectrolyte (i.e., polyacrylamide, PAM) concentration, specifically its aging, of the salt concentration is shown. The close agreement of the technique with classical bulk rheological measurements is demonstrated, illustrating the advantages of the technique.

A novel chemotaxis assay in 3-D collagen gels by time-lapse microscopy.

The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.

Microfluidics analysis of red blood cell membrane viscoelasticity.

In this work, a microfluidic system to investigate the flow behavior of red blood cells in a microcirculation-mimicking network of PDMS microchannels with thickness comparable to cell size is presented. We provide the first quantitative description of cell velocity and shape as a function of the applied pressure drop in such devices. Based on these results, a novel methodology to measure cell membrane viscoelastic properties in converging/diverging flow is developed, and the results are in good agreement with data from the literature. In particular, in the diverging channel the effect of RBC surface viscosity is dominant with respect to shear elasticity. Possible applications include measurements of cell deformability in pathological samples, where reliable methods are still lacking.

Droplet deformation under confined Poiseuille flow.

The flow behavior of droplet-based liquid-liquid systems, such as emulsions, polymer blends, and foodstuff, which are ubiquitous in everyday life, has attracted scientific interest in different disciplines. In this review, we focus on the pressure-driven confined flow behavior of isolated droplets in circular and rectangular cross-section channels, which are valuable model geometries to gain insight into more complex flow conditions found in industrial applications. The effect of the relevant nondimensional parameters governing droplet deformation and breakup, such as viscosity ratio, capillary number, and ratio of droplet to tube radius, is presented both for axisymmetric and off-axis droplets, including cross-stream migration. The role of surfactants is also discussed. Ongoing research directions include the field of microfluidics techniques, where confined flow geometries can be exploited to manipulate droplets with a variety of possible applications.

A methodology to study the deformability of red blood cells flowing in microcapillaries in vitro.

The deformability of red blood cells flowing in microvessels is essential to maintain optimal blood circulation and to allow gas transfer between blood and tissues. Here, we report on an experimental methodology to investigate the deformability of RBCs flowing in microcapillaries having diameter close to the average cell size. The microcapillaries are placed in a rectangular flow cell, where a suspension of RBCs, properly diluted in albumin-additioned ACD, is fed through a syringe under the action of a liquid head in the physiological range. Video microscopy images of the flowing RBCs are acquired at high magnification and later processed by an automated image analysis macro. It was found that RBCs from healthy donors exhibit the classical parachute shape observed in vivo. Furthermore, all the data of healthy RBC velocity vs liquid head are well represented by the same linear regression, independently on the donor. Preliminary results on beta-thalassemia RBCs are also presented and show, on the average, a reduced velocity compared to healthy samples.