Fetal nucleated red blood cell analysis for non-invasive prenatal diagnostics using a nanostructure microchip.

Cell-free DNA has been widely used in non-invasive prenatal diagnostics (NIPD) nowadays. Compared to these incomplete and multi-source DNA fragments, fetal nucleated red blood cells (fNRBCs), once as an aided biomarker to monitor potential fetal pathological conditions, have re-attracted research interest in NIPD because of their definite fetal source and the total genetic information contained in the nuclei. Isolating these fetal cells from maternal peripheral blood and subsequent cell-based bio-analysis make maximal genetic diagnosis possible, while causing minimal harm to the fetus or its mother. In this paper, an affinity microchip is reported which uses hydroxyapatite/chitosan nanoparticles as well as immuno-agent anti-CD147 to effectively isolate fNRBCs from maternal peripheral blood, and on-chip biomedical analysis was demonstrated as a proof of concept for NIPD based on fNRBCs. Tens of fNRBCs can be isolated from 1 mL of peripheral blood (almost 25 mL-1 in average) from normal pregnant women (from the 10th to 30th gestational week). The diagnostic application of fNRBCs for fetal chromosome disorders (Trisomy 13 and 21) was also demonstrated. Our method offers effective isolation and accurate analysis of fNRBCs to implement comprehensive NIPD and to enhance insights into fetal cell development.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Surface Acoustic Waves Grant Superior Spatial Control of Cells Embedded in Hydrogel Fibers.

By exploiting surface acoustic waves and a coupling layer technique, cells are patterned within a photosensitive hydrogel fiber to mimic physiological cell arrangement in tissues. The aligned cell-polymer matrix is polymerized with short exposure to UV light and the fiber is extracted. These patterned cell fibers are manipulated into simple and complex architectures, demonstrating feasibility for tissue-engineering applications.

On-Chip Production of Size-Controllable Liquid Metal Microdroplets Using Acoustic Waves.

Micro- to nanosized droplets of liquid metals, such as eutectic gallium indium (EGaIn) and Galinstan, have been used for developing a variety of applications in flexible electronics, sensors, catalysts, and drug delivery systems. Currently used methods for producing micro- to nanosized droplets of such liquid metals possess one or several drawbacks, including the lack in ability to control the size of the produced droplets, mass produce droplets, produce smaller droplet sizes, and miniaturize the system. Here, a novel method is introduced using acoustic wave-induced forces for on-chip production of EGaIn liquid-metal microdroplets with controllable size. The size distribution of liquid metal microdroplets is tuned by controlling the interfacial tension of the metal using either electrochemistry or electrocapillarity in the acoustic field. The developed platform is then used for heavy metal ion detection utilizing the produced liquid metal microdroplets as the working electrode. It is also demonstrated that a significant enhancement of the sensing performance is achieved by introducing acoustic streaming during the electrochemical experiments. The demonstrated technique can be used for developing liquid-metal-based systems for a wide range of applications.

Three-dimensional manipulation of single cells using surface acoustic waves.

The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving "acoustic tweezers" in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner.

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.

BACKGROUND: Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. METHODS AND FINDINGS: We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. CONCLUSIONS: Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.