Gradient elution moving boundary electrophoresis for use with complex samples and detection of toxins
Inventors
Henry, Alyssa Catharyn • Ross, David Judson
Assignees
Applied Research Associates Inc • National Institute of Standards and Technology NIST
Publication Number
US-11479804-B1
Publication Date
2022-10-25
Expiration Date
2030-04-16
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Abstract
Methods of detecting the presence of toxins in a sample using electrophoretic separations and of performing electrophoretic separation of complex samples are provided. The method of detecting the presence of toxins includes reacting a sample and a substrate with a signaling enzyme which converts the substrate to the product in a reaction medium, introducing a run buffer into a separation channel having an inlet end, selectively introducing at least one of the substrate and the product of the reaction medium into the inlet end of the separation channel, electrophoretically separating the substrate and the product, and determining the rate of conversion of the substrate to the product, wherein a change in the rate of conversion is indicative of the presence of toxins. The method of performing electrophoretic separations of complex samples having charged particulates and oppositely charged analytes comprising introducing a run buffer into a separation channel having an inlet end, selectively introducing the oppositely charged analytes in the complex sample into the separation channel, and electrophoretically separating the charged particulates and the oppositely charged analytes. Additionally, a device for varying with respect to time the bulk flow of a fluid in a separation channel of an electrophoretic device having a buffer reservoir in fluid contact with the separation channel is provided. The device includes a pressure sensor in fluid contact with a buffer reservoir, a high pressure reservoir in selective fluidic communication with the buffer reservoir, a low pressure reservoir in selective fluidic communication with the buffer reservoir and in fluidic communication with the high pressure reservoir, and a pumping device for pumping a gas from the low pressure reservoir to the high pressure reservoir.
Core Innovation
The invention provides methods of detecting toxins in a sample using electrophoretic separations and performing electrophoretic separations of complex samples. The method for detecting toxins involves reacting a sample and a substrate with a signaling enzyme which converts the substrate to a product in a reaction medium. The reaction mixture is introduced into a separation channel with a run buffer, where substrate and product are electrophoretically separated by applying an electric potential and varying the bulk flow over time. The rate of conversion of substrate to product is determined, where changes in this rate indicate the presence of toxins.
The invention also provides a method for electrophoretic separation of complex samples containing charged particulates and oppositely charged analytes. Run buffer is introduced into a separation channel, the oppositely charged analytes are selectively introduced, and electrophoretic separation is performed by applying an electric potential and varying the bulk flow in a direction substantially aligned with the electric potential. The analytes are sequentially detected and quantified.
Additionally, the invention describes a device for varying the bulk flow of fluid in a separation channel of an electrophoretic device having a buffer reservoir in fluid contact with the channel. This device includes a pressure sensor in contact with the buffer reservoir, high and low pressure reservoirs in selective fluid communication with the buffer reservoir, and a pumping device for transferring gas from the low to the high pressure reservoir. The device operates in three conditions to vary pressure in the buffer reservoir and thus variable bulk flow over time during electrophoretic separation.
Claims Coverage
The patent includes one independent claim directed to a device for varying the bulk flow of fluid in a separation channel and its main inventive features are detailed below.
Device structure for controlling bulk fluid flow
A device comprising a pressure sensor in fluid contact with a buffer reservoir that is in selective venting communication with the atmosphere, a high pressure reservoir selectively fluidically connected to the buffer reservoir, a low pressure reservoir selectively fluidically connected to the buffer reservoir and fluidically connected to the high pressure reservoir, and a pumping device transferring gas from the low to the high pressure reservoir.
Three operating conditions for pressure regulation
The device operates through three conditions: (1) high pressure reservoir in fluid communication with the buffer reservoir to reach a threshold pressure level (5,000 Pa to 60,000 Pa), (2) buffer reservoir in fluid communication with the low pressure reservoir to initiate a pressure ramp varying pressure from a starting to an ending value, and (3) high pressure reservoir again in fluid communication with the buffer reservoir to reach a final pressure level (5,000 Pa to 60,000 Pa).
Selective valves and flow resistors for pressure and venting control
Control valves provided in fluidic communication with the buffer, high pressure, and low pressure reservoirs allow selective fluid communication, often together with flow resistors to regulate the rate of pressure changes and venting to the ambient atmosphere.
Use of check valves and pump placement
A pair of check valves are disposed between the low and high pressure reservoirs, with the pumping device positioned between these check valves to direct gas flow and maintain pressure differentials.
Rigid outer casing for device packaging
The device may include a rigid outer casing that houses the reservoirs, valves, sensors, and flow regulating components to form an integrated structure.
Pressure ramp specifications
The starting pressure value is about 10,000 Pa and the ending pressure value about -10,000 Pa, with the pressure varied at a rate of approximately -300 Pa/s during the pressure ramp.
Overall, the independent claim protects a multi-reservoir and valve-controlled device for precisely varying fluid bulk flow by orchestrating pressure changes via a pumping device and controlled fluid communication, enabling pressure ramps and set pressure thresholds within an electrophoretic separation system.
Stated Advantages
Allows detection of toxins in complex samples with minimal sample preparation.
Enables electrophoretic separations without sample injection or moving parts, providing high data quality over short separation lengths.
Permits indirect detection of toxins by monitoring enzyme activity through substrate-product conversion rate.
Facilitates sequential separation and quantification of substrates, products, charged particulates, and analytes via controlled variable bulk flow.
Provides a portable, automated device to precisely vary bulk fluid flow via pressure control for improved separation performance.
Documented Applications
Detection of toxins such as nerve agents, pesticides, and endotoxins in various samples by measuring enzyme activity changes.
Electrophoretic separation and quantification of ionic species and charged analytes in complex matrices including soil, mud, milk, apple juice, estuarine sediment, coal fly ash, blood serum, and plant leaves.
Field-deployable toxin detection in situ due to portable device design.
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