JP-7855181-B2 - Microfluidic particle-labeled impedance sensor array for enhancing bioassay sensitivity

JP7855181B2JP 7855181 B2JP7855181 B2JP 7855181B2JP-7855181-B2

Inventors

キム，ジュンキュ
バビル，アリ・コダヤリ

Assignees

ユニバーシティオブユタリサーチファウンデーション
テキサス・テック・ユニバーシティー・システム

Dates

Publication Date: 20260508
Application Date: 20220310
Priority Date: 20210312

Claims (20)

A microfluidic device for detecting and quantifying biomolecules, (a) A multilayer microfluidic network comprising a microfluidic channel, a buffer inlet, a sample inlet, and a waste outlet, wherein the buffer inlet, sample inlet, and waste outlet are fluidly connected to each other via the microfluidic channel, and the microfluidic network is configured to accept a buffer solution and a sample. A substrate layer comprising a surface to which a first antibody is covalently bound, adapted so that the first antibody specifically binds to a target analyte, and the first antibody is mounted between a first electrode and a second electrode, wherein the first electrode and the second electrode are part of a plurality of electrodes, the plurality of electrodes are combined with each other, and the distance between the first electrode and the second electrode is 1 μm to 10 μm. The substrate layer is in fluid communication with a microfluidic channel, and the first antibody is located within the microfluidic channel. Microfluidic chips and (b) A detector for detecting changes in electrical impedance, A device that includes this.
The device according to claim 1, wherein the microfluidic network is composed of a polymer material selected from one of the following: polycarbonate (PC), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), polyimide, polydimethylsiloxane (PDMS), or a combination thereof.
The device according to claim 2, wherein the microfluidic network is composed of PDMS.
The device according to claim 1, wherein the microfluidic network has geometric dimensions for autonomous capillary flow of the buffer solution and sample.
The device according to claim 4, wherein the microfluidic network comprises a chamber layer, a capillary valve, and a bridging hole located above the microfluidic channel, the chamber layer and the microfluidic channel are in fluid communication with each other via the capillary valve and the bridging hole, and the sample inlet is located within the chamber layer.
The device according to claim 5, wherein the chamber layer contains a second antibody, the second antibody is adapted to specifically bind to a target analyte, and the second antibody is conjugated to microparticles.
The device according to claim 6, wherein the second antibody comprises a biotin moiety, the microparticle comprises a streptavidin coating, and the second antibody is conjugated to the microparticle through the binding of the biotin moiety to the streptavidin coating.
The device according to claim 6, wherein the fine particles include magnetic beads, polystyrene beads, silica beads, or a combination thereof.
The device according to claim 8, wherein the fine particles have a size in the range of 1 μm to 5 μm in diameter.
The device according to claim 9, wherein the fine particles include magnetic beads having a diameter of 2.8 μm.
The device according to claim 6, wherein the chamber layer further comprises a porous polycarbonate (PC) membrane connected to the sample inlet, and microparticles conjugated to the second antibody are immobilized on the porous PC membrane.
The device according to claim 5, wherein the capillary valve comprises an orifice having a size in the range of 100 μm to 300 μm in diameter .
The device according to claim 12, wherein the capillary valve comprises an orifice having a diameter of 250 μm.
The device according to claim 5, wherein the bridging hole comprises an orifice having a size in the range of 0.5 mm to 2.5 mm in diameter .
The device according to claim 14, wherein the bridging hole comprises an orifice having a diameter of 1 mm.
The device according to claim 1, wherein the buffer solution contains phosphate-buffered saline (PBS) at a concentration in the range of 0.001 mM to 1 mM.
The device according to claim 16, wherein the buffer solution contains PBS at a concentration of 0.01 mM.
The device according to claim 16, wherein the buffer solution further comprises 1 wt% bovine serum albumin (BSA).
The device according to claim 1, wherein the microfluidic network further comprises one or more absorbent pads.
The device according to claim 1, wherein the sample inlet further comprises a serum separation membrane.

Description

Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63/160,594, filed on 12 March 2021, which is incorporated herein by reference as is. This research was funded by the federal government. This invention was made with government support under authorization number NSF-EECS/1509746 granted by the National Science Foundation. The government has certain rights in this invention. This specification describes improved devices and methods for the detection and quantification of biomolecules. The devices and methods may include a microfluidic biosensing platform for measuring changes in electrical impedance in the presence of a target analyte from a sinusoidal input voltage to improve the sensitivity of immunoassays. In one embodiment, the described devices and methods may provide a point-of-care immunoassay platform for quantitative diagnostics that uses signal-enhanced microparticles to improve sensitivity and detection limits. Impedance-measuring biosensors are a class of electrochemical biosensors that measure changes in electrical impedance caused by the presence of biomolecules, cells, or labeled biomaterials on the working electrode by applying a sinusoidal voltage. Due to their low cost, ease of miniaturization, multiplexing capability, and label-free operation, these types of sensors have proven promising for digitized point-of-care (POC) diagnostics. To date, considerable effort has been made, in conjunction with the development of miniaturized platforms with reduced complexity. Previous research has led to the development of an integrated cell-counting assay system for malaria diagnosis, consisting of a microfluidic chip and a miniature impedance circuit board. Another integrated diagnostic platform, consisting of a printed gold electrode chip and a microfluidic flow cell, has been proposed for the detection of the transgenic protein Cry1Ab. For a miniaturized portable impedance-measuring biosensor platform, an impedance-measuring reader based on the AD5933 chip has been designed using microfluidic channels on an IDE array for the diagnosis of deep vein thrombosis and pulmonary embolism. Despite considerable effort in developing miniaturized impedance biosensors, few integrated platforms with sufficient sensitivity and ease of use for actual immunoassays have been developed. Capillary microfluidic technology has been used to perform different types of immunoassays on different platforms. In some of the earliest studies, the potential of on-chip immunoassays using the capillary filling phenomenon was investigated. To obtain an autonomous fluid pathway, a capillary holding valve was introduced to allow for sequential delivery and multiple cycles. By controlling the geometric shape of the channel, and therefore the hydrodynamic resistance, a one-step capillary-driven microfluidic system was developed that enabled flow velocity control and varied incubation time within the reaction chamber. This compact device demonstrated a quantitative immunoassay for C-reactive protein (CRP), with the lowest detectable fluorescence signal corresponding to CRP at 10 pg/mL. While such platforms paved the way for autonomous microfluidic chips for performing highly sensitive immunoassays, their low sensitivity and the need for large, bulky downstream sensors to read the output signal hindered their development into commercially viable products. This figure shows a schematic diagram of an exemplary microfluidic impedance sensor array for microparticle-labeled immunoassays. It shows eight interconnected electrodes (IDEs) integrated with a specially designed impedance analyzer and microfluidic channels (left). Illustrative diagrams of the IDEs against negative controls (top) and positive controls (bottom) are shown, and the formation of complete immune complexes on the IDEs is illustrated (right).This figure shows an exemplary system configuration consisting of a gold (Au) IDE array chip, two impedance analysis circuits, and a LabVIEW software program and associated data acquisition (DAQ) board for signal transduction. A microfluidic channel network may be located at the top of the IDE array to deliver analytes and buffer solutions, and to control fluid forces for washing purposes.This figure shows a schematic diagram of an exemplary IDE chip fabrication process.This diagram illustrates the IDE and its equivalent circuit, as well as the equations that show how the IDE measures impedance fluctuations from an applied sinusoidal voltage and a measured alternating current.This graph shows the gain coefficients calculated for different frequencies based on measurements taken using an impedance analyzer and an LCR meter in PBS buffers of different concentrations.This figure shows the equivalent circuit of an impedance biosensor for particulate labeling immunoassays. The equivalent circuit consists of the capacitance effect of the electrodes, the double-layer capacitance o