This invention relates generally to detection assays and, more particularly, to a device and method incorporating fluidics, bead-based enzymatic assays, and/or fluorescence for target detection, such as cholesterol detection.
Microfluidics based tissue engineering mimics the physiological environment of tissues and organs. Perfusion is one process mimicked by microfluidics; however, continuous measurement of cellular secretion requires the analysis to be on-line. One approach for on-line analysis includes enzymatic assay on-a-chip using mixing channels for reagents and sample
One of the benefits of the microfluidic tissue systems is that the cells can be subjected to physiological shear stress by perfusion. However, this brings upon a challenge in determining cellular function because on-line or continuous analysis of secretory components in very limited. Moreover, these secretions are generated in low volumes which further makes it challenging for analysis. There is a continuing need for improved microfluidic-based assays.
A general object of the invention is to provide an improved detection assay. The general object of the invention can be attained, at least in part, through a method of and apparatus for detecting components in a microfluidic sample.
The method of embodiments of this invention include: providing the microfluidic sample in a microfluidic device; introducing a plurality of microbeads to the sample within the microfluidic device, wherein each of the microbeads comprises a plurality of bioactive proteins, molecules, lipoproteins, or combinations thereof, bound thereon; generating a fluorescent signal from a reaction of the microbeads and the microfluidic sample; and detecting or measuring the generated fluorescent signal.
In embodiments of this invention the microfluidic device comprises mixing channels. In embodiments of this invention the microbeads comprise polystyrene beads. In embodiments of this invention the bioactive protein comprises streptavidin or an enzyme.
In embodiments of this invention the method includes generating a fluorescent signal by generating peroxide, wherein an intensity of fluorescence is directly proportional to a concentration of the peroxide generated. In embodiments of this invention generating a fluorescent signal includes oxidizing a fluorogenic material. Desirably the signal is detected and measured in real-time or near real-time. In embodiments of this invention the signal is detected intermittently or continuously for a determined period of time, without replacing or replenishing reagents.
Other objects and advantages will be apparent to those skilled in the art from the following detailed description taken in conjunction with the appended claims and drawings.
A general object of the invention is to provide an assay system or method, such as an enzymatic assay, to detect components in the low volume of microfluidic samples. Microfluidics enables precise engineering of microphysiological environments for in vitro mimicking of tissues and organs. These ‘organs-on-a-chip’ beneficially use lower number of cells and lower quantities of reagents than conventional processes.
Embodiments of this invention include a bead-based enzymatic assay ‘on-a-chip’. The use of beads (e.g., 20 nm to 200 μm in diameter) for the assay minimizes the assay volume and addresses the problem of the conventional assays requiring high volumes of samples and reagents. The invention provides an assay that is simpler than current techniques and is very versatile to be used in all enzyme based assays, such as for glucose, glycerol, triglyceride, and/or cholesterol. In embodiments of the invention, the polystyrene bead is bound with streptavidin, and used to immobilize the enzyme via biotin.
The enzymatic assay of this invention can incorporate detection by fluorescence or other suitable detection means. The invention measures target composition levels by reading the fluorescent signal generated by an analyte and the enzymes using laser excitation and a photomultiplier tube. In bead-based assays, standard fluorescent microscopy can be used to capture the individual fluorescent beads. The images can be analyzed, such as using ImageJ, to quantify the target, such as cholesterol secreted by the cells.
Embodiments of this invention include a bead-based enzymatic assay with horseradish peroxidase (HRP) enzyme immobilized on the polystyrene beads. As shown in
The invention provides benefits over conventional solution based enzymatic assays, in particular because: the conventional assays use a microplate reader for detection of signal and these cannot be adapted to other analytical formats such as fluorescence microscopy or flow cytometry, and continuous real-time monitoring of biomolecules such as cholesterol is not possible for cell based or biological applications using conventional assays, typically because the medium or the sample needs to be collected before running the assay.
Bead-based enzymatic assays provide the fluorescent signal localized on a solid substrate, e.g., a polystyrene bead, making the assay adaptable to different formats such as fluorescence microscopy, flow cytometry, and microplate reader. It is possible to monitor secretions of biomolecules such as cholesterol in real-time using continuous collection of samples or medium and running the assay. For example, samples from perfusion based microfluidic hepatocyte cultures or liver-on-a-chip can be used with the bead-based enzymatic assay for real-time monitoring of cholesterol.
Oxidation reaction involving HRP generates the fluorescent signal on the bead as HRP is immobilized on the bead. Visual and image analysis is possible because of localized fluorescent signal. Real-time continuous monitoring of biomolecules secreted by the perfusion based microfluidic cultures and organs-on-a-chip is possible as the fluorescent signal on the beads can be detected and quantified using fluorescent microscopy and image analysis. Unlike the conventional enzymatic assays which are suitable for detection by microplate readers only, the bead-based enzymatic assay can be used in microplate readers, and also with fluorescence microscopy as well as flow cytometry.
The system and method of this invention are useful for detection of biomolecules such as hydrogen peroxide, cholesterol, glycerol, glucose, pyruvate, or any such molecules which use the oxidation reaction of resazurin to resorufin catalyzed by HRP for signal generation in biological samples using detection methods such as microplate reader, fluorescence microscopy, or flow cytometry. The system and method can be used to study cellular or biological response in the form of secretion of biomolecules such as hydrogen peroxide, cholesterol, glycerol, glucose, pyruvate, etc., to different biochemical stimuli using microplate readers or fluorescence microscope for detecting the signal. The system and method can be used in cytometric bead array format in flow cytometry for assessing the enzymatic or oxidative activity
The bead-based enzymatic assay for the chemistry of oxidation reaction of resazurin to resorufin catalyzed by HRP was tested using the enzyme immobilized beads and hydrogen peroxide standards.
The assay has also been used to measure cholesterol secreted from liver hepatoma cells that are cultured on a microfluidic device (
Thus, the invention provides an enzymatic assay to detect components in low volume of microfluidic samples. The bead-based enzymatic assay on-a-chip can be used to detect cholesterol in low volume of microfluidic samples.
The invention illustratively disclosed herein suitably may be practiced in the absence of any element, part, step, component, or ingredient which is not specifically disclosed herein.
While in the foregoing detailed description this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
This application claims the benefit of U.S. Provisional Application, Ser. No. 62/627,381, filed on 7 Feb. 2018. The co-pending Provisional Application is hereby incorporated by reference herein in its entirety and is made a part hereof, including but not limited to those portions which specifically appear hereinafter.
This invention was made with government support under contract 7R00DK095984-04 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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62627381 | Feb 2018 | US |