NanoDLD Chip for Phosphoprotein Purification and Detection
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Solution Overview
Problem
Current methods for detecting and quantifying phosphoproteins are limited by sensitivity, specificity, and the ability to perform single-cell and single-molecule analysis, with existing techniques often requiring large sample volumes, fixation, or struggling with ionization of phosphate groups.
Innovation Solution
A chip-based pillar array using nanoscale deterministic lateral displacement (nanoDLD) for size-based separation and charge separation, combined with fluorescently-labeled antibodies or antibody-coated beads, to effectively purify and quantify phosphoproteins by forming complexes that exceed the size threshold, allowing for single-molecule detection and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to detect phosphoproteins, then sensitivity can be achieved, but phosphorylated proteins are difficult to ionize and detect over background peaks
Solution Approach 1:
The patent introduces an intermediary step of immunoprecipitation using phospho-specific antibodies before mass spectrometry analysis. This mediator selectively enriches phosphoproteins from complex samples, forming antibody-protein complexes that can be purified and concentrated, thereby overcoming the ionization difficulty and background interference issues in direct mass spectrometry detection
2Measurement precision
If ELISA is used for quantitative detection, then sensitivity is improved, but the volumes required do not allow for single-cell studies
Solution Approach 1:
The patent segments the detection process into two stages: first, immunoprecipitation in a small volume to capture phosphoproteins from single cells; second, pooling multiple immunoprecipitates to achieve sufficient quantity for quantitative analysis. This segmentation allows maintaining single-cell resolution while accumulating enough material for accurate quantification
3Quantity of substance
If flow cytometry is used for single-cell analysis, then cell-to-cell heterogeneity can be detected, but single-molecule detection and quantification within each cell is not possible
Solution Approach 1:
The patent implements a nested approach where immunoprecipitation (molecular-level purification) is nested within flow cytometry (single-cell analysis). By performing immunoprecipitation on individually sorted cells and then analyzing the purified phosphoproteins, the method achieves both single-cell resolution and single-molecule detection sensitivity
4Quantity of substance
If immunofluorescence is used to detect cell-to-cell heterogeneity, then spatial information is preserved, but sensitivity and antibody performance are frequent issues
Solution Approach 1:
The patent applies preliminary immunoprecipitation to enrich and concentrate phosphoproteins before detection. By performing this preliminary purification and concentration step, the sensitivity of subsequent detection methods is dramatically improved, overcoming the sensitivity limitations of direct immunofluorescence while preserving cell-to-cell heterogeneity information
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables robust, quantitative detection and purification of phosphoproteins, overcoming limitations of existing methods by allowing single-cell analysis and high-sensitivity assays without ionization issues, and effectively separating phosphorylated proteins from non-phosphorylated counterparts.
Implementation Method 1
nanoscale deterministic lateral displacement (nanoDLD) array having a plurality of pillars separated by a gap g, wherein the antibody and proteins in the protein sample form antibody-protein complexes having a size that is greater than a size threshold of the nanoDLD array created by the gap g which permits size-based separation
Data Source
AI summary
Techniques for phosphoprotein detection, quantification, and purification using a chip-based pillar array are provided. In one aspect, a method for purifying a protein sample includes: introducing a mixture including the protein sample and an antibody to a nanoDLD array having a plurality of pillars separated by a gap g, wherein the antibody and proteins in the protein sample form antibody-protein complexes having a size that is greater than a size threshold of the nanoDLD array created by the gap g which permits size-based separation of the antibody-protein complexes as the mixture flows through the nanoDLD array; and collecting a purified protein sample containing the antibody-protein complexes from the nanoDLD array. A lab-on-a-chip (LOC) device including the nanoDLD array is also provided.