Rapid Quantification of Plasma Creatinine Using a Novel Kinetic Enzymatic Assay.

BACKGROUND: Enzymatic assays are among the most common diagnostic tests performed in the clinical laboratory. Enzymatic substrate analysis is most commonly measured using endpoint methods; however, modulating the reaction kinetics allows fine control of the reaction rate, which can be adjusted based on specific monitoring technologies. METHODS: We developed and optimized an enzymatic method for measurement of creatinine in plasma, using commonly paired enzymes of creatininase (Crtnnase), creatinase (Crtase), sarcosine oxidase (SOX), ascorbate oxidase (AOX), and horseradish peroxidase (HRP). The novel aspect of the assay is that it is fast and uses SOX as the limiting enzyme. The assay performance was assessed with respect to precision, accuracy, and interferences. RESULTS: The intrarun %CV (n = 12) was approximately 5% for each concentration tested, with biases ranging from -3 to -9%. The interrun %CV (n = 39) ranged from 5 to 8%, with biases ranging from -2 to -6%. During the accuracy assessment (n = 127), only 4 samples did not meet the minimum acceptability criteria. Minimal interference was observed, except at low creatinine concentrations with elevated creatine. CONCLUSION: Our novel and versatile enzymatic assay to measure plasma creatinine using kinetic analysis with SOX as the limiting enzyme is rapid (<2 mins), sensitive, and specific and demonstrates excellent concordance with the laboratory standard. We anticipate this rapid kinetic assay to be compatible with emerging technologies in the field of portable diagnostic devices, such as the usage of silicon photonics to monitor biochemical reactions.

Generative optical modeling of whole blood for detecting platelets in lens-free images.

In this paper, we consider the task of detecting platelets in images of diluted whole blood taken with a lens-free microscope. Despite having several advantages over traditional microscopes, lens-free imaging systems have the significant challenge that the resolution of the system is typically limited by the pixel dimensions of the image sensor. As a result of this limited resolution, detecting platelets is very difficult even by manual inspection of the images due to the fact that platelets occupy just a few pixels of the reconstructed image. To address this challenge, we develop an optical model of diluted whole blood to generate physically realistic simulated holograms suitable for training machine learning models in a supervised manner. We then use this model to train a convolutional neural network (CNN) for platelet detection and validate our approach by developing a novel optical configuration which allows collecting both lens-free and fluorescent microscopy images of the same field of view of diluted whole blood samples with fluorescently labeled platelets.

Development of nanostars as a biocompatible tumor contrast agent: toward in vivo SERS imaging.

The need for sensitive imaging techniques to detect tumor cells is an important issue in cancer diagnosis and therapy. Surface-enhanced Raman scattering (SERS), realized by chemisorption of compounds suitable for Raman spectroscopy onto gold nanoparticles, is a new method for detecting a tumor. As a proof of concept, we studied the use of biocompatible gold nanostars as sensitive SERS contrast agents targeting an ovarian cancer cell line (SKOV3). Due to a high intracellular uptake of gold nanostars after 6 hours of exposure, they could be detected and located with SERS. Using these nanostars for passive targeting after systemic injection in a xenograft mouse model, a detectable signal was measured in the tumor and liver in vivo. These signals were confirmed by ex vivo SERS measurements and darkfield microscopy. In this study, we established SERS nanostars as a highly sensitive contrast agent for tumor detection, which opens the potential for their use as a theranostic agent against cancer.

Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.

Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 µm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.

Tailored and biodegradable poly(2-oxazoline) microbeads as 3D matrices for stem cell culture in regenerative therapies.

We present the synthesis of hydrogel microbeads based on telechelic poly(2-oxazoline) (POx) crosslinkers and the methacrylate monomers (HEMA, METAC, SPMA) by inverse emulsion polymerization. While in batch experiments only irregular and ill-defined beads were obtained, the preparation in a microfluidic (MF) device resulted in highly defined hydrogel microbeads. Variation of the MF parameters allowed to control the microbead diameter from 50 to 500 µm. Microbead elasticity could be tuned from 2 to 20 kPa by the POx:monomer composition, the POx chain length, net charge of the hydrogel introduced via the monomer as well as by the organic content of the aqueous phase. The proliferations of human mesenchymal stem cells (hMSCs) on the microbeads were studied. While neutral, hydrophilic POx-PHEMA beads were bioinert, excessive colonization of hMSCs on charged POx-PMETAC and POx-PSPMA was observed. The number of proliferated cells scaled roughly linear with the METAC or SPMA comonomer content. Additional collagen I coating further improved the stem cell proliferation. Finally, a first POx-based system for the preparation of biodegradable hydrogel microcarriers is described and evaluated for stem cell culturing.

Poly(2-oxazoline)-Based Microgel Particles for Neuronal Cell Culture.

An increasing number of in vivo and in vitro neuro-engineering applications are making use of colloidal particles as neuronal cell carriers. Recent studies highlight the shortcomings of commercial glass particles and stress the benefit of using soft microgel particles (MGPs) instead. This study describes first the fabrication of MGPs from telechelic poly(2-methyl-2-oxazoline)s (PMeOx) cross-linkers and hydrophilic neutral (hydroxyethyl)methacrylate (HEMA) or charged 2-methacryloxyethyltrimethylammonium (METAC) monomers by emulsion polymerization, and it discusses their ability to support cell growth. It establishes that uncharged copolymers lead to MGPs with nonfouling properties inappropriate for cell culture, and it provides a protocol to amend their surface properties to enable cell adhesion. Finally, it demonstrates that the introduction of positive charges by METAC is necessary to obtain surface properties suitable for neuronal cell development. Through the optimization of the PMeOx30 MGP properties, this work provides general guidelines to evaluate and tune MGP chemistry to obtain microcarriers for neuro-engineering applications.