Cell Membrane Quantitation via Reference Signal Correction
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Solution Overview
Problem
Current methods for quantifying biological materials on cell membranes in pathological samples face challenges due to variations in protein denaturation and sample preparation, leading to inaccurate results, especially when using fluorescent labeling techniques, as they fail to specifically isolate the target protein signal from non-specifically adsorbed signals and are influenced by the degraded state of samples.
Innovation Solution
A method involving multiple immunostaining using fluorescent labels for both the target biological material and reference biological materials, where the target material is labeled with fluorescent dye-containing nanoparticles and the reference material with fluorescent dyes, allowing for accurate quantification by correcting the signal using the reference material's fluorescence intensity, thereby isolating the target material's signal on the cell membrane.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If fluorescent labeling techniques are used to quantify target biological material on cell membranes, then the ability to detect and measure fluorescence signal is improved, but the measurement precision deteriorates due to non-specific adsorption and sample degradation variations
Solution Approach 1:
The patent introduces a reference biological material as an intermediary standard that mediates the quantification process. This reference material is stained with a fluorescent label and serves as a control to correct for variations in sample preparation, fixation, and detection conditions. By comparing the target material's fluorescence signal against the reference material's signal, the method eliminates the need for absolute quantification and compensates for non-specific adsorption and degradation effects.
Solution Approach 2:
The patent changes the quantification approach from absolute fluorescence intensity measurement to relative ratio measurement. Instead of measuring the absolute fluorescence signal of the target biological material alone, the method calculates the ratio between the target material's fluorescence signal and the reference material's fluorescence signal. This parameter transformation converts the measurement from an absolute value susceptible to environmental variations into a relative value that is inherently corrected for those variations.
2Measurement precision
If multiple immunostaining with fluorescent labels is performed, then the ability to specifically identify target protein location is improved, but the device complexity and procedure complexity increase
Solution Approach 1:
The patent employs a universal reference biological material that can be stained alongside various target materials using the same procedural framework. The reference material serves multiple functions: it corrects for sample preparation variations, provides a normalization standard, and enables comparison across different target proteins and experimental conditions. This multi-functionality reduces the need for separate control experiments and simplifies the overall workflow despite the use of multiple fluorescent labels.
3Stability of the object's composition
If fluorescent labels are used instead of enzyme markers, then the stability of the marker signal is improved, but the reliability of quantification deteriorates due to non-specific binding and sample condition variations
Solution Approach 1:
The patent implements a feedback mechanism where the reference biological material's fluorescence signal provides real-time information about the effectiveness of sample preparation and staining conditions. By comparing the target material signal with the reference material signal, the system automatically compensates for variations in fixation quality, permeabilization efficiency, and fluorescent label performance. This feedback loop ensures reliable quantification even when sample conditions vary.
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
This approach enhances the accuracy of quantifying target biological materials on cell membranes, improving the reliability of pathological diagnosis and the assessment of molecular target drug effectiveness by minimizing the impact of sample preparation variations and non-specific binding.
Implementation Method 1
fluorescent labeling techniques using fluorescent labels instead of enzyme markers and capable of allowing antigens to appear as bright spots of fluorescence observable in the dark field (fluorescence microscope or confocal laser microscope)
Data Source
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Figure 1B
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AI summary
The present invention provides a method capable of more accurately quantifying a biological material expressed on the cell membrane in pathological samples. The present invention is directed to a method for quantifying a biological material (target biological material) expressed on the cell membrane, the method including the steps of: (1a) immunostaining the target biological material with a fluorescent material; (1b) immunostaining another biological material (reference biological material) on the cell membrane with another fluorescent material; (2) using immunostaining images for the target and reference biological materials to identify the fluorescence signal corresponding to the target biological material and to measure the fluorescence signals corresponding to the target and reference biological materials; and (3) correcting the measured value of the fluorescence signal corresponding to the target biological material by a given method to quantify the expression level.