US-12623218-B2 - Well array device, system and methods of use thereof

Abstract

The microfluidic chip and the microfluidic system of the present invention provides a unique integration of a microfluidic chip and a label-free quantification process. The microfluidic chip uses well arrays and dielectrophoresis (DEP) to capture a polarizable agent in a well. Once the polarizable agents have been captured, non-faradaic electrochemical impedance spectroscopy (nF-EIS) measurements can be performed to quantify the polarizable agent.

Inventors

• Jie Chen
• Lukas Menze
• Pedro A. Duarte

Assignees

• Hidaca Ltd.

Dates

Publication Date

20260512

Application Date

20211217

Claims (18)

1. 1 . A microfluidic chip for selecting and detecting a polarizable agent in a sample containing a mixture of such agents, comprising: a substrate, at least three individually addressable electrodes on the substrate with a gap between adjacent electrodes, forming a plurality of gaps, a well array on the substrate having a plurality of wells positioned on the plurality of gaps, each well located on a single gap, a top layer on the well array, and a microfluidic channel, between the top layer and the well array, wherein the microfluidic chip comprises at least 2 sectors, the at least three individually addressable electrodes include two electrodes in each sector, with different sectors optionally sharing one of the two electrodes, and each sector comprises different multiple wells of the plurality of wells.
2. 2 . The microfluidic chip of claim 1 , wherein the at least three individually addressable electrodes comprise a metal selected from the group consisting of: aluminum, gold, copper, carbon, titanium, silver, platinum, palladium and mixtures thereof.
3. 3 . The microfluidic chip of claim 1 , wherein the well array is formed in a material selected from the group consisting of: epoxy, hydrogel, polydimethylsiloxane, polyurethane and polyethylene glycol.
4. 4 . The microfluidic chip of claim 1 , wherein the plurality of wells are circular and each has a diameter of at most 30 μm.
5. 5 . The microfluidic chip of claim 1 , wherein the well array comprises 1,000 to 10,000 wells.
6. 6 . The microfluidic chip of claim 1 , wherein the depth of each of the plurality of wells is 5 to 20 μm, and the width or diameter of each of the plurality of wells is 5 to 50 μm.
7. 7 . A microfluidic system for analyzing a liquid sample containing a mixture of polarizable agents, comprising: the microfluidic chip of claim 1 , further comprising an input lumen and an output lumen fluidly connected to the microfluidic channel, an input fluid tube, fluidly connected to the input lumen of the microfluidic chip, an output fluid tube, fluidly connected to the output lumen of the microfluidic chip, a controller, and connectors, electrically connecting the at least three individually addressable electrodes of the microfluidic chip to the controller, wherein the controller is configured to, with the two electrodes in each sector: (i) generate a different dielectrophoresis (DEP) force in each sector, and (ii) measure an impedance in each sector.
8. 8 . The microfluidic system of claim 7 , wherein the microfluidic system is configured to measure the impedance using non-faradaic electrochemical impedance spectroscopy (nF-EIS).
9. 9 . The microfluidic system of claim 7 , further comprising a pump to introduce the liquid sample into the input fluid tube.
10. 10 . The microfluidic system of claim 7 , wherein the controller comprises: a CPU, a memory, a function generator, an oscilloscope, and an impedance analyzer.
11. 11 . The microfluidic system of claim 7 , wherein the connectors comprise a chip holder.
12. 12 . The microfluidic system of claim 7 , wherein the controller further comprises a computer program stored in a memory to operate the microfluidic system.
13. 13 . A method of making the microfluidic system of claim 7 , comprising: forming the microfluidic chip, forming the at least three individually addressable electrodes on the substrate, forming the well array on the substrate, connecting the at least three individually addressable electrodes to the controller, wherein the controller comprises a function generator and an impedance analyzer.
14. 14 . The microfluidic system of claim 7 , wherein the controller determines the concentration of each of the polarizable agents present in the liquid sample.
15. 15 . The microfluidic chip of claim 1 , wherein: the well array comprises 100 to 10,000 wells, and the at least 2 sectors comprises 2 to 20 sectors.
16. 16 . A microfluidic system for analyzing a liquid sample containing a mixture of polarizable agents, comprising: (a) a microfluidic chip, including: (i) a substrate, (ii) at least three individually addressable electrodes on the substrate with a gap between adjacent electrodes, forming a plurality of gaps, (iii) a well array on the substrate having a plurality of wells positioned on the plurality of gaps, each well located on a single gap, (iv) a top layer on the well array, (v) a microfluidic channel, between the top layer and the well array, (vi) an input lumen and (vii) an output lumen fluidly connected to the microfluidic channel, wherein the microfluidic chip comprises 2 to 20 sectors, each sector comprising different multiple wells of the plurality of the wells, and the at least three individually addressable electrodes include two electrodes in each sector, with different sectors optionally sharing one of the two electrodes, (b) an input fluid tube, fluidly connected to the input lumen of the microfluidic chip, (c) an output fluid tube, fluidly connected to the output lumen of the microfluidic chip, (d) a controller, and (e) connectors, electrically connecting the at least three individually addressable electrodes of the microfluidic chip to the controller, wherein the controller is configured to, with the two electrodes in each sector: (i) generate a different dielectrophoresis (DEP) force in each sector, and (ii) measure an impedance in each sector.
17. 17 . The microfluidic system of claim 16 , wherein the depth of each of the plurality of wells is 5 to 20 μm, and the width or diameter of each of the plurality of wells is 5 to 50 μm.
18. 18 . The microfluidic system of claim 16 , wherein the well array comprises 100 to 10,000 wells.

Description

BACKGROUND Single-cell sorting is of increasing importance in precision medicine applications such as advanced genomics or next generation sequencing.6-8 State-of-the-art systems for cancer detection, such as CellSearch,9,10 OncoDiscover,11 or CellMax,12,13 capture and isolate circulating tumor cells (CTCs) from patient samples. Subsequent quantification of the number of CTCs then may provide important information about the state of a patient. In single-cell RNA sequencing platforms, such as in the commercially available 10× Genomics solutions14, 15 or other recent advances,16-18 it is crucial to ensure that only the target cells are sequenced for accurate results. Hence, depending on the mechanism of the sequencing platform, reliable cell sorting prior to loading the sequencing platform may be required. Traditionally used cell sorting approaches include density gradient-based methods as well as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). In both FACS and MACS, the sample has to be prepared and correctly labeled with either fluorescent stains or magnetic microbeads in order to be sorted successfully.1,4 Dielectrophoresis activated cell sorting (DACS) offers an alternative approach based on the intrinsic characteristics of dielectrophoresis (DEP) to sort cells without the need for any prior labeling. DEP is an electrokinetic phenomenon that allows for the selective manipulation of dielectric agents, such as mammalian cells, via polarization when subjected to a spatially nonuniform electric field.19 Reported microfluidic DACS structures mainly focus on sorting cells by positioning electrodes in a microfluidic channel and subsequently use DEP to change the flow trajectory of passing cells depending on their dielectric characteristics, hence directing them into different output channels.20-25 These designs are innately limited by the number of output channels, as the number of individually sorted cell types directly corresponds to the number of outlets. Other less common DEP-based cell sorting approaches employ field-flow fractionation to sort different cell types.26,27 Another limitation these designs face is their inability to readily provide information about the number of sorted cells. Additional cell counting structures such as microfluidic Coulter counters, which have been shown to reliably detect single cells,28-30 may be employed to count the number of cells flowing through an outlet channel at the cost of an increased overall system complexity. Infectious plant diseases caused by microorganisms such as fungi and bacteria are one of the main factors affecting crop production, resulting in huge economic losses to farmers and growers.64-66 Among the list of numerous diseases, Sclerotinia stem rot (SSR) is of particular importance due to its wide host range and harmful effects.67-68 SSR is caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, affecting more than 400 plant species worldwide, including several economically important crops such as canola, soybean, sunflower, and carrot.67-69,70 SSR, also commonly known as white mold, is particularly devastating to the canola industry, the world's second-largest oilseed crop.71 Yield losses due to SSR can be as high as 50%, causing severe financial losses72 and making it the greatest threat to canola production. Microscopic spores produced by the fungus disseminate throughout the fields in wind currents, representing the primary source of inoculum initiating SSR epidemics.69,73 Currently, chemical control employing fungicides is the main strategy for the management of SSR.74 Although this approach can be highly effective, fungicides are economically inefficient when applied routinely and with no indication of disease risk. Ideally, farmers must apply fungicides during specific time frames and only when necessary, that is, when spores are present in the field but before symptoms are visible.74,75 However, as SSR outbreaks are hard to predict, farmers typically apply fungicides routinely and without any objective information on the risk of SSR development, a decision that costs time, drastically reduces the profits, and affects the environment. Current methods for predicting SSR development are imprecise. Risk assessment checklists76-77 and weather-based forecasting models78-80 were the first systems developed for this purpose. Although simple and field-specific, the checklists are time-consuming, labor-intensive, and do not include any measurement of airborne inoculum. On the other hand, weather-based systems lack field specificity and are based exclusively on weather parameters. SUMMARY In a first aspect, the present invention is a microfluidic chip for selecting and detecting a polarizable agent in a sample containing a mixture of such agents, including: a substrate, at least three electrodes on the substrate with a gap between adjacent electrodes, forming a plurality of gaps, a well array on the subst