Microfluidic technology has potential to separate sperm cells from unwanted debris while improving the effectiveness of assisted reproductive technologies (ART). Current clinical protocol limitations regarding the separation of sperm cells from other cells/cellular debris can lead to low sperm recovery when the sample contains a low concentration of mostly low motility sperm cells and a high concentration of unwanted cells/cellular debris, such as in semen samples from patients with pyospermia [high white blood cell (WBC) semen]. This study demonstrates label-free separation of sperm cells from such semen samples using inertial microfluidics. The approach does not require any externally applied forces except the movement of the fluid sample through the instrument. Using this approach, it was possible to recover not only any motile sperm, but also viable less-motile and non-motile sperm cells with high recovery rates. Our results demonstrate the ability of inertial microfluidics to significantly reduce WBC concentration by flow focusing of target WBCs within a spiral channel flow. The estimated sample process time was more rapid (∼5 min) and autonomous than the conventional method (gradient centrifuge sperm wash; ∼1 h). A mixture of sperm/WBC was injected as the device input and 83% of sperm cells and 93% of WBCs were collected separately from two distinct outlets. The results show promise for enhancing sperm samples through inertial flow processing of WBCs and sperm cells that can provide an advantage to ART procedures such as sample preparation for intrauterine insemination.

Microfluidic sperm sorting has historically relied on sperm motility. However, a motility-based sperm separation technology will not work when viable, non-motile sperm need to be separated from other tissues as occurs when performing testicular sperm extraction (TESE) and microdissection testicular sperm extraction (mTESE) techniques. This work demonstrates the use of inertial microfluidics technology using spiral channels to separate sperm from blood cells. The separation method, which is label-free, does not rely on sperm motility for sorting. Basic principles of spiral channel separations were used to design a specific channel and flow parameters for separating non-motile sperm from blood. The spiral channels dimensions were: initial radius, 0.7 cm; final radius, 0.899 cm; channel width, 150 μm; channel height, 50 μm; turns of spiral, 4 turns; and space between channels, 310 μm. If sperm are modeled as a 5 μm sphere, inertial microfluidics theory suggests that the sperm could be focused and separated from red blood cells (RBCs). Channels to implement these features were validated in a series of experiments. Mixed samples of RBCs and sperm were used to test the sperm separation capability of the device with the sample injection flow rate ranging from 0.1–0.52 ml min⁻¹. After running the sample through the spiral channel, the samples were collected from four outlets and were inspected using microscopy. The best results were obtained at a 0.52 ml min⁻¹ flow rate and generated a concentration ratio of 81%, representing the percent of sperm collected from the two outer outlets. For the same conditions, 99% of RBCs were collected from the two inner wall outlets. Using a high speed scanner, we were able to observe the focusing of the RBCs and general focusing of the sperm. As the sperm are not a uniform shape, they did not focus in a tight band, but were collected in a general region of the channel. Nevertheless, the purification ratio for these sperm was sufficient to greatly enhance the likelihood of finding rare sperm in TESE/mTESE samples containing millions of blood cells. Sequentially processing of the samples in the system proved to further improve the ratio of sperm to blood cells.

Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Plant growth-promoting rhizobacteria (PGPR) play a crucial role in biological control and pathogenic defense on and within plant tissues, however the mechanisms by which plants associate with PGPR to elicit such beneficial effects need further study. Here, we present time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of Brachypodium distachyon (Brachypodium) seeds with and without exposure to two model PGPR, i.e., Gram-negative Pseudomonas fluorescens SBW25 (P.) and Gram-positive Arthrobacter chlorophenolicus A6 (A.). Delayed image extraction was used to image PGPR-treated seed sections to reveal morphological changes. ToF-SIMS spectral comparison, principal component analysis (PCA), and two-dimensional (2D) imaging show that the selected PGPR have different effects on the host seed surface, resulting in changes in chemical composition and morphology. Metabolite products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and indole-3-acetic acid (IAA), were identified on the PGPR-treated seed surfaces. These compounds have different distributions on the Brachypodium seed surface for the two PGPR, indicating that the different bacteria elicit distinct responses from the host. Our results illustrate that ToF-SIMS is an effective tool to study plant–microbe interactions and to provide insightful information with submicrometer lateral resolution of the chemical distributions associated with morphological features, potentially offering a new way to study the mechanisms underlying beneficial roles of PGPR.