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-9261482-B1
Publication Date
2016-02-16
Expiration Date
2030-04-16
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 the presence of toxins in a sample using electrophoretic separations, specifically employing enzymatic assays coupled with gradient elution moving boundary electrophoresis (GEMBE). The method involves reacting a sample with a signaling enzyme that converts a substrate to a product in a reaction medium. The substrate and product are then selectively introduced into a separation channel filled with a run buffer, electrophoretically separated under an applied electric potential while varying the bulk flow of the run buffer over time, and sequentially detected and quantified. Changes in the rate of conversion of substrate to product indicate the presence of toxins in the sample.
Further, the invention addresses the problem of performing electrophoretic separations on complex samples containing charged particulates and oppositely charged analytes without extensive sample preparation. By introducing the complex sample into a separation channel with run buffer and applying GEMBE, oppositely charged analytes are selectively introduced, electrophoretically separated, and sequentially detected while varying the bulk flow of the run buffer in alignment with the electric potential.
The background identifies a significant need for toxin detection methods that are portable, sensitive at low concentrations, and capable of analyzing complex, unfiltered samples. Existing methods like ion mobility spectrometry and capillary electrophoresis have limitations including bulkiness and sample preparation requirements. GEMBE offers advantages such as no need for sample injection, no moving parts, short separation lengths, and high data quality. The invention also includes a novel device for varying the bulk flow over time in the electrophoretic device, thereby controlling sample introduction and separation.
Claims Coverage
The patent contains multiple independent claims focused on methods of performing GEMBE to separate complex samples and the related inventive features.
Method for GEMBE separation of complex samples with minimal or no sample preparation
Introducing a run buffer and a complex sample into a separation channel; employing GEMBE by applying an electric potential along the channel; varying the bulk flow of run buffer with time in a direction aligned with the electric potential; selectively introducing oppositely charged analytes into the channel for sequential detection and quantification; wherein the bulk flow comprises electroosmotic flow combined with controlled, variable pressure-driven flow.
Method for GEMBE separation of unfiltered complex samples
Introducing a run buffer into a separation channel with defined inlet (sample reservoir) and outlet (buffer reservoir); introducing an unfiltered complex sample into the sample reservoir; applying an electric potential to effect electrophoretic migration of oppositely charged analytes; varying the bulk flow over time in alignment with the electric potential to selectively introduce the analytes for sequential detection and quantification.
Method for GEMBE separation of prepared complex samples
Preparing a complex sample by dilution or suspension; introducing a run buffer and the sample into a separation channel; applying an electric potential along the channel; varying the bulk flow of run buffer over time aligned with the electric potential; selectively introducing and electrophoretically separating the oppositely charged analytes for sequential detection and quantification.
The claims focus on the inventive use of GEMBE with controlled variable bulk flow, including combined electroosmotic and pressure-driven flow, applied to complex, unfiltered samples with minimal preparation, enabling selective sequential electrophoretic separation and detection of oppositely charged analytes amidst charged particulates.
Stated Advantages
GEMBE does not require sample injection or moving parts, providing electrophoretic separations over short lengths with high data quality.
The invention enables detection of toxins at lower concentrations than permitted by traditional conductivity detection methods.
The method allows for electrophoretic separation of complex samples with little or no sample preparation, avoiding issues caused by particulates entering separation capillaries such as noisy signals or clogging.
The described device enables controlled, time-varied bulk flow in the separation channel, enhancing resolution and selectivity of electrophoretic separations.
Documented Applications
Detection of toxins including nerve agents and pesticides (e.g., organophosphates) by indirect enzymatic assays using acetylcholinesterase or adenylate cyclase in complex samples such as soil, mud, dirt, milk, apple juice, estuarine sediment, coal fly ash, tomato leaves, peach leaves, citrus leaves, and calf serum.
Analysis and separation of charged particulates and oppositely charged analytes in complex environmental and biological samples without prior filtration or extensive preparation.
Use of the invented device for controlling bulk flow to perform GEMBE separations with improved resolution and sequential detection of substrate and product in enzymatic assays and of analytes in complex samples.
Interested in licensing this patent?