Toward point-of-care diagnostics: Running enzymatic assays on a photonic waveguide-based sensor chip with a portable, benchtop measurement system.

We demonstrate a portable, compact system to perform absorption-based enzymatic assays at a visible wavelength of 639 nm on a photonic waveguide-based sensor chip, suitable for lab-on-a-chip applications. The photonic design and fabrication of the sensor are described, and a detailed overview of the portable measurement system is presented. In this publication, we use an integrated photonic waveguide-based absorbance sensor to run a full enzymatic assay. An assay to detect creatinine in plasma is simultaneously performed on both the photonic sensor on the portable setup and on a commercial microplate reader for a clinically relevant creatinine concentration range. We observed a high correlation between the measured waveguide propagation loss and the optical density measurement from the plate reader and measured a limit-of-detection of 4.5 µM creatinine in the sensor well, covering the relevant clinical range for creatinine detection.

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.

Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion.

Cancer cell invasion from primary tumors is mediated by a complex interplay between cellular adhesions, actomyosin-driven contractility, and the physical characteristics of the extracellular matrix (ECM). Here, we incorporate a mechanochemical free-energy-based approach to elucidate how the two-way feedback loop between cell contractility (induced by the activity of chemomechanical interactions such as Ca2+ and Rho signaling pathways) and matrix fiber realignment and strain stiffening enables the cells to polarize and develop contractile forces to break free from the tumor spheroids and invade into the ECM. Interestingly, through this computational model, we are able to identify a critical stiffness that is required by the matrix to break intercellular adhesions and initiate cell invasion. Also, by considering the kinetics of the cell movement, our model predicts a biphasic invasiveness with respect to the stiffness of the matrix. These predictions are validated by analyzing the invasion of melanoma cells in collagen matrices of varying concentration. Our model also predicts a positive correlation between the elongated morphology of the invading cells and the alignment of fibers in the matrix, suggesting that cell polarization is directly proportional to the stiffness and alignment of the matrix. In contrast, cells in nonfibrous matrices are found to be rounded and not polarized, underscoring the key role played by the nonlinear mechanics of fibrous matrices. Importantly, our model shows that mechanical principles mediated by the contractility of the cells and the nonlinearity of the ECM behavior play a crucial role in determining the phenotype of the cell invasion.

Collective cancer cell invasion induced by coordinated contractile stresses.

The physical underpinnings of fibrosarcoma cell dissemination from a tumor in a surrounding collagen-rich matrix are poorly understood. Here we show that a tumor spheroid embedded in a 3D collagen matrix exerts large contractile forces on the matrix before invasion. Cell invasion is accompanied by complex spatially and temporally dependent patterns of cell migration within and at the surface of the spheroids that are fundamentally different from migratory patterns of individual fibrosarcoma cells homogeneously distributed in the same type of matrix. Cells display a continuous transition from a round morphology at the spheroid core, to highly aligned elongated morphology at the spheroid periphery, which depends on both ß1-integrin-based cell-matrix adhesion and myosin II/ROCK-based cell contractility. This isotropic-to-anisotropic transition corresponds to a shift in migration, from a slow and unpolarized movement at the core, to a fast, polarized and persistent one at the periphery. Our results also show that the ensuing collective invasion of fibrosarcoma cells is induced by anisotropic contractile stresses exerted on the surrounding matrix.