The rapid and accurate analysis of micro-samples is a crucial foundation for precision medicine, particularly for early screening and monitoring of cancer, where it holds significant importance. Ultrasound-based multifunctional biocompatible manipulation techniques have been extensively applied in a variety of biomedical fields, providing insights for the development of rapid, cost-effective, and accurate biomarker detection strategies. In this chapter, we combine ultrasound-based gradient pressure fields with functionalized microsphere enrichment to develop a biosensing method for ultra-trace miRNA enrichment in nanoliter samples without PCR. This system relies on inexpensive capillaries, enabling simultaneous visual imaging and trace sample detection.

The resolving power, chemical sensitivity and non-invasive nature of NMR have made it an established technique for in vivo studies of large organisms both for research and clinical applications. NMR would clearly be beneficial for analysis of entities at the microscopic scale of about 1 nL (the nanoliter scale), typical of early development of mammalian embryos, microtissues and organoids: the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 µm) and sensitivity issues have prevented a widespread adoption of NMR. In this article we show how these limitations can be overcome to obtain NMR spectra of a mammalian embryo in its early stage. To achieve this we employ ultra-compact micro-chip technologies in combination with 3D-printed micro-structures. Such device is packaged for use as plug & play sensor and it shows sufficient sensitivity to resolve NMR signals from individual bovine pre-implantation embryos. The embryos in this study are obtained through In Vitro Fertilization (IVF) techniques, transported cryopreserved to the NMR laboratory, and measured shortly after thawing. In less than 1 h these spherical samples of just 130-190 µm produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectra vary from one sample to another despite their optical and morphological similarities, suggesting that the method can further develop into a non-invasive embryo assay for selection prior to embryo transfer.

\"Children are not tiny adults\" is an adage commonly used in pediatrics to emphasize the fact that children often have different physiological responses to sickness and trauma compared to adults. However, despite widespread acceptance of this concept, diagnostic blood testing is an excellent example of clinical care that is not yet customized to the needs of children, especially newborns. Cumulative blood loss resulting from clinical testing does not typically impact critically ill adult patients, but can quickly escalate in children, leading to iatrogenic anemia and related comorbidities. Moreover, the tests prioritized for rapid, near-patient testing in adults are not always the most clinically relevant tests for children or newborns. This report describes the development of a digital microfluidic testing platform and associated clinical assays purposely curated to address current shortcomings in pediatric laboratory testing by using microliter volumes (<50 µL) of samples. The automated platform consists of a small instrument and single-use cartridges, which contain all reagents necessary to prepare the sample and perform the assay. Electrowetting technology is used to precisely manipulate nanoliter-sized droplets of samples and reagents inside the cartridge. To date, we have automated three disparate types of assays (biochemical assays, immunoassays, and molecular assays) on the platform and have developed over two dozen unique tests, each with important clinical application to newborns and pediatric patients. Cell lysis, plasma preparation, magnetic bead washing, thermocycling, incubation, and many other essential functions were all performed on the cartridge without any user intervention. The resulting assays demonstrate performance comparable to standard clinical laboratory assays and are economical due to the reduced hands-on effort required for each assay and lower overall reagent consumption. These capabilities allow a wide range of assays to be run simultaneously on the same cartridge using significantly reduced sample volumes with results in minutes.

Droplets of aqueous solutions distributed in an immiscible oil phase are increasingly used and investigated as a means to handle and assay small volumes of samples. The primary attraction of this method is that surface interactions are kept to a minimum, and changes in sample concentration, especially due to adsorption to the walls, are avoided. Microfluidic methods to generate, transport, merge, split and perform reactions in droplets were developed recently. These methods depend on the continuous flow of the two phases involved inside closed microfluidic channels. Alternatively, an electrowetting phenomenon was also exploited to control the movement of droplets between two solid substrates. However, there are some situations where small volume sample transport and assaying are required in open systems. Here, we demonstrate a simple electromechanical probe (tweezers) that is capable of manipulating a small aqueous droplet in a bi-layer oil phase. The tweezer consists of two needles positioned close to each other and uses polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. Mechanical motion of the tweezer can be used to transport the droplet to various positions. Operations such as aliquoting, merging and transport are demonstrated. Finally, this method was used to perform a DNA amplification assay where droplets of the sample and the amplification mixture are aliquoted separately, mixed and amplified using an in-situ heater. This electromechanical tweezer is of interest in low-throughput, small-volume biological and chemical assays where the investigator requires direct and open access to the samples.

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy.