Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing.

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models.

Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1.

Exposure to polystyrene nanoplastics reduces bacterial and fungal biomass in microfabricated soil models.

Nanoplastics have been proven to induce toxicity in diverse organisms, yet their effect on soil microbes like bacteria and fungi remains largely unexplored. In this paper, we used micro-engineered soil models to investigate the effect of polystyrene (PS) nanospheres on Pseudomonas putida and Coprinopsis cinerea. Specifically, we explored the effects of increasing concentrations of 60 nm carboxylated bovine serum albumin (BSA) coated nanospheres (0, 0.5, 2, and 10 mg/L) on these bacterial and fungal model organisms respectively, over time. We found that both microorganisms could disperse through the PS solution, but long-distance dispersal was reduced by high concentrations. Microbial biomass decreased in all treatments, in which bacteria showed a linear dose response with the strongest effect at 10 mg/L concentration, and fungi showed a non-linear response with the strongest effect at 2 mg/L concentration. At the highest nanoplastics concentration, the first colonizing fungal hyphae adsorbed most of the PS nanospheres present in their vicinity, in a process that we termed the 'vacuum cleaner effect'. As a result, the toxicity effect of the original treatment on subsequently growing fungal hyphae was reduced to a growth level indistinguishable from the control. We did not find evidence that nanoplastics are able to penetrate bacterial nor fungal cell walls. Overall, our findings provide evidence that nanoplastics can cause a direct negative effect on soil microbes and highlight the need for further studies that can explain how the microbial stress response might affect soil functions.

Habitat complexity affects microbial growth in fractal maze.

The great variety of earth's microorganisms and their functions are attributed to the heterogeneity of their habitats, but our understanding of the impact of this heterogeneity on microbes is limited at the microscale. In this study, we tested how a gradient of spatial habitat complexity in the form of fractal mazes influenced the growth, substrate degradation, and interactions of the bacterial strain Pseudomonas putida and the fungal strain Coprinopsis cinerea. These strains responded in opposite ways: complex habitats strongly reduced fungal growth but, in contrast, increased the abundance of bacteria. Fungal hyphae did not reach far into the mazes and forced bacteria to grow in deeper regions. Bacterial substrate degradation strongly increased with habitat complexity, even more than bacterial biomass, up to an optimal depth, while the most remote parts of the mazes showed both decreased biomass and substrate degradation. These results suggest an increase in enzymatic activity in confined spaces, where areas may experience enhanced microbial activity and resource use efficiency. Very remote spaces showing a slower turnover of substrates illustrate a mechanism which may contribute to the long-term storage of organic matter in soils. We demonstrate here that the sole effect of spatial microstructures affects microbial growth and substrate degradation, leading to differences in local microscale spatial availability. These differences might add up to considerable changes in nutrient cycling at the macroscale, such as contributing to soil organic carbon storage.

Habitat geometry in artificial microstructure affects bacterial and fungal growth, interactions, and substrate degradation.

Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order. Sharper angles reduced fungal and bacterial biomass, especially when angles were repeated in the same direction. Substrate degradation was only decreased by sharper angles when fungi and bacteria were grown together. Investigation at the cellular scale suggests that this was caused by fungal habitat modification, since hyphae branched in sharp and repeated turns, blocking the dispersal of bacteria and the substrate. Our results demonstrate how the geometry of microstructures can influence microbial activity. This can be transferable to soil pore spaces, where spatial occlusion and microbial feedback on microstructures is thought to explain organic matter stabilization.

Microfluidic chips provide visual access to in situ soil ecology.

Microbes govern most soil functions, but investigation of these processes at the scale of their cells has been difficult to accomplish. Here we incubate microfabricated, transparent 'soil chips' with soil, or bury them directly in the field. Both soil microbes and minerals enter the chips, which enables us to investigate diverse community interdependences, such as inter-kingdom and food-web interactions, and feedbacks between microbes and the pore space microstructures. The presence of hyphae ('fungal highways') strongly and frequently increases the dispersal range and abundance of water-dwelling organisms such as bacteria and protists across air pockets. Physical forces such as water movements, but also organisms and especially fungi form new microhabitats by altering the pore space architecture and distribution of soil minerals in the chip. We show that soil chips hold a large potential for studying in-situ microbial interactions and soil functions, and to interconnect field microbial ecology with laboratory experiments.

Acoustophoresis in polymer-based microfluidic devices: Modeling and experimental validation.

A finite-element model is presented for numerical simulation in three dimensions of acoustophoresis of suspended microparticles in a microchannel embedded in a polymer chip and driven by an attached piezoelectric transducer at MHz frequencies. In accordance with the recently introduced principle of whole-system ultrasound resonances, an optimal resonance mode is identified that is related to an acoustic resonance of the combined transducer-chip-channel system and not to the conventional pressure half-wave resonance of the microchannel. The acoustophoretic action in the microchannel is of comparable quality and strength to conventional silicon-glass or pure glass devices. The numerical predictions are validated by acoustic focusing experiments on 5-µm-diameter polystyrene particles suspended inside a microchannel, which was milled into a polymethylmethacrylate chip. The system was driven anti-symmetrically by a piezoelectric transducer, driven by a 30-V peak-to-peak alternating voltage in the range from 0.5 to 2.5 MHz, leading to acoustic energy densities of 13 J/m3 and particle focusing times of 6.6 s.

Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.

How do fungi navigate through the complex microscopic maze-like structures found in the soil? Fungal behaviour, especially at the hyphal scale, is largely unknown and challenging to study in natural habitats such as the opaque soil matrix. We monitored hyphal growth behaviour and strategies of seven Basidiomycete litter decomposing species in a micro-fabricated "Soil Chip" system that simulates principal aspects of the soil pore space and its micro-spatial heterogeneity. The hyphae were faced with micrometre constrictions, sharp turns and protruding obstacles, and the species examined were found to have profoundly different responses in terms of foraging range and persistence, spatial exploration and ability to pass obstacles. Hyphal behaviour was not predictable solely based on ecological assumptions, and our results obtained a level of trait information at the hyphal scale that cannot be fully explained using classical concepts of space exploration and exploitation such as the phalanx/guerrilla strategies. Instead, we propose a multivariate trait analysis, acknowledging the complex trade-offs and microscale strategies that fungal mycelia exhibit. Our results provide novel insights about hyphal behaviour, as well as an additional understanding of fungal habitat colonisation, their foraging strategies and niche partitioning in the soil environment.

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate.

Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics.

Acoustic impedance matched buffers enable separation of bacteria from blood cells at high cell concentrations.

Sepsis is a common and often deadly systemic response to an infection, usually caused by bacteria. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality. To replace the time-consuming blood culture we are developing a method to directly separate bacteria from red and white blood cells to enable faster bacteria identification. The blood cells are moved from the sample flow into a parallel stream using acoustophoresis. Due to their smaller size, the bacteria are not affected by the acoustic field and therefore remain in the blood plasma flow and can be directed to a separate outlet. When optimizing for sample throughput, 1 ml of undiluted whole blood equivalent can be processed within 12.5 min, while maintaining the bacteria recovery at 90% and the blood cell removal above 99%. That makes this the fastest label-free microfluidic continuous flow method per channel to separate bacteria from blood with high bacteria recovery (>80%). The high throughput was achieved by matching the acoustic impedance of the parallel stream to that of the blood sample, to avoid that acoustic forces relocate the fluid streams.

Acoustofluidic hematocrit determination.

Hematocrit (HCT) measurements of blood from patients, blood donors and athletes are routinely performed on a daily basis. These measurements are often performed in centralized hospital labs by whole blood analyzers, which leads to long time-to-result. On site measurements, based on centrifugation can be done, but these assays require manual handling, are slow and can just measure HCT in contrast to the central lab whole blood analyzers. In this work, we present a microfluidic based method to measure HCT in blood samples by acoustic separation of whole blood into discrete regions of plasma and red blood cells. Comparison of the areas of the red blood cell and plasma regions gives an accurate HCT value, with a linear correlation to the centrifugation-based reference method. A readout can be performed within 2 s of acoustic actuation providing a readout accuracy of approximately 3% points (pp) HCT. Additional accuracy can be achieved by extending the acoustic actuation to 20 s, yielding an error of less than 1 pp HCT. This acoustic tool is optimal for integration into a lab-on-a-chip device with in-line measurements of different clinical parameters.

Build your own soil: exploring microfluidics to create microbial habitat structures.

Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis.

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 µl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.

Integrated Acoustic Separation, Enrichment, and Microchip Polymerase Chain Reaction Detection of Bacteria from Blood for Rapid Sepsis Diagnostics.

This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.

Microchip electroseparation of proteins using lipid-based nanoparticles.

Porous liquid crystalline lipid-based nanoparticles are shown here to enable protein analysis in microchip electroseparation by reducing sample adsorption. Additionally, higher stability and reproducibility of the separations were observed. The method was tested by separating green fluorescent protein (GFP) in hot embossed cyclic olefin polymer microchips with integrated fiber grooves for LIF detection. The sample adsorption was indirectly quantified by measuring the height, width and asymmetry of the separation peaks for various concentrations of nanoparticles in the sample and background electrolyte. Without nanoparticles, electropherograms displayed typical signs of extensive adsorption to the channel walls, with low, broad tailing peaks. Higher, narrower more symmetric peaks were generated when 0.5-10% nanoparticles were added, showing a dramatic reduction of sample adsorption. The current through the separation channel decreased with nanoparticle concentration, reducing to half its value when the nanoparticle concentration was increased from 0.5 to 4%. Addition of nanoparticles enabled separations that were otherwise hindered by extensive adsorption, e.g. separation of GFP mutants differing by only one amino acid. It was also observed that increasing the nanoparticle concentration increased the number of impurities that could be resolved in a GFP sample. This indicates that the adsorption is further reduced, and/or that the nanoparticles provide an interacting pseudostationary phase for electrochromatography.

Electrophoresis microchip with integrated waveguides for simultaneous native UV fluorescence and absorbance detection.

Simultaneous label-free detection of UV absorbance and native UV-excited fluorescence in an electrophoresis microchip is presented. UV transparent integrated waveguides launch light at a wavelength of 254 nm from a mercury lamp along the length of a 1-mm long detection cell. Transmitted UV light is collected by another waveguide in the opposite end of the detection cell, while visible fluorescence is collected vertically through the lid of the chip. The background of scattered excitation light is suppressed by detection perpendicular to the excitation, the limited UV transparency of the borosilicate lid and by choosing a PMT insensitive to the excitation light. This way, the need for a fluorescence filter is eliminated. Calibration curves were measured for serotonin, tryptophan, propranolol and acetaminophen, and separations of the four compounds were demonstrated by electrophoresis and MEKC. All compounds could be detected in the micromolar range by absorbance detection, but fluorescence detection improved detection limits for compounds displaying native UV fluorescence up to ten times. The simultaneous detection also proved useful for the identification of compounds with similar retention times and even enables accurate quantification of co-eluting compounds.