Multiplexed Homocysteine and Cysteine Screening by Chemical Reduction
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Solution Overview
Problem
Current first-tier newborn screening assays for homocystinuria (HCU) face challenges due to the complex chemistry of homocysteine (Hcy), leading to high false positive and false negative rates, necessitating costly and time-consuming second-tier screening, and existing multiplexing methods are limited by on-chip electrophoresis devices with low throughput.
Innovation Solution
A method involving the use of reducing agents like TCEP and thiol derivatizing agents like N-ethyl maleimide (NEM) to convert Hcy and cysteine (Cys) forms into monomers, followed by derivatization and mass spectrometry analysis without prior chromatographic separation, allowing simultaneous quantitation of total Hcy and cysteine in a single assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If methionine is used as a surrogate marker for HCU screening in first-tier assays, then the assay can be performed with existing methodology, but false positive and false negative rates increase and nearly 50% of HCU cases are missed
Solution Approach 1:
The patent changes the chemical form of homocysteine from dimer/protein complex to monomer through reduction, enabling direct detection. This parameter change (molecular structure) allows Hcy to be detected as a free amino acid rather than through surrogate markers, resolving the contradiction between using existing methodology and achieving accurate detection
Solution Approach 2:
The patent introduces a reducing agent as an intermediary substance that converts Hcy dimers and protein complexes into monomers. This intermediary enables the detection of total Hcy (tHcy) directly, eliminating the need for methionine surrogate markers and improving both reliability and measurement precision
2Measurement precision
If second-tier screening assay with separation step is used to test for total Hcy, then detection accuracy improves, but analysis time increases, additional costs are incurred, and capital equipment is required
Solution Approach 1:
The patent extracts the essential function of second-tier screening (tHcy detection) and brings it into the first-tier assay through chemical reduction. By removing the need for separate separation steps and capital equipment, the invention achieves accurate tHcy detection in the rapid first-tier screening workflow
Solution Approach 2:
The patent performs preliminary chemical reduction of Hcy dimers and protein complexes to monomers before mass spectrometry analysis. This preliminary action enables direct detection of total Hcy in the first-tier assay, eliminating the need for subsequent second-tier separation steps and reducing overall screening time
3Reliability
If on-chip electrophoresis device is used for multiplexing Hcy and Cys, then first-tier screening of Hcy is achieved, but throughput is reduced and device complexity increases
Solution Approach 1:
The patent replaces the mechanical electrophoresis separation system with a chemical reduction approach followed by direct mass spectrometry analysis. This substitution eliminates the need for complex on-chip electrophoresis devices while maintaining the ability to multiplex Hcy and Cys detection, thereby preserving throughput
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 and throughput of HCU screening, reducing false positives and negatives, eliminating the need for second-tier screening, and maintaining the stability of co-analyzed biomarkers, with analysis times under 3 minutes per sample.
Implementation Method 1
contacting a blood sample with a reducing agent optionally in the presence of a solvent to convert at least one of a Hcy dimer, a Hcy-protein complex, a Cys dimer different from the Hcy dimer, or a Cys-protein complex in the blood sample to a Hcy monomer, a Cys monomer, or a combination thereof
Implementation Method 2
reacting the Hcy monomer, the Cys monomer, or a combination thereof in the monomer sample with a thiol derivatizing agent to form a thiol derivatized sample comprising a thiol derivatized Hcy monomer, a thiol derivatized Cys monomer
Implementation Method 3
analyzing the thiol derivatized sample, the thiol-and-ester derivatized sample, or a ready-to-use sample with mass spectrometry to quantify a level of tHcy, a level tCys
Data Source
AI summary
A method of multiplexing Hcy and/or Cys in a first-tier screening assay includes: contacting a blood sample with a reducing agent optionally in the presence of a solvent to convert at least one of a Hcy dimer, a Hcy-protein complex, a Cys dimer different from the homocysteine dimer, or a Cys-protein complex in the blood sample to a Hcy and/or Cys monomer thereby forming a monomer sample; reacting the Hcy and/or Cys monomer in the monomer sample with a thiol derivatizing agent to form a thiol derivatized sample comprising a thiol derivatized Hcy and/or Cys monomer; and optionally converting a carboxylic acid or a carboxylate group in the thiol derivatized Hcy and/or Cys monomer to an ester, forming a thiol-and-ester derivatized sample comprising a thiol-and-ester derivatized Hcy and/or Cys monomer. The method can be used to determine a level of tHcy, a level of tCys, and/or a level of CysT in a blood sample, and screen for CBS deficiency, HCU, or hyperhomocysteinemia.