Upconversion Cross-Correlation Spectroscopy of a Sandwich Immunoassay

Area of Science:

• Biophysics
• Analytical Chemistry
• Biotechnology

Background:

• Fluorescence Correlation Spectroscopy (FCS) and Cross-Correlation Spectroscopy (FCCS) are powerful tools for studying biological processes in living cells.
• However, strong autofluorescence in biological samples like plasma complicates FCS/FCCS analysis.
• Existing methods struggle to distinguish true fluorescence signals from background noise in complex biological matrices.

Purpose of the Study:

• To introduce a novel spectroscopic technique, Upconversion Cross-Correlation Spectroscopy (UCCS), for analyzing biological samples.
• To demonstrate UCCS's capability to eliminate autofluorescence interference in biological fluids.
• To validate UCCS for quantitative immunoassays in challenging matrices like plasma.

Main Methods:

• Utilized two distinct upconversion nanoparticles (UCNPs) emitting at different wavelengths.
• Employed a single excitation laser positioned on the anti-Stokes side.
• Implemented UCCS to measure fluorescence signals, specifically targeting Stokes-shifted autofluorescence removal.
• Applied UCCS to a homogeneous sandwich immunoassay for thyroid-stimulating hormone (TSH).

Main Results:

• UCCS effectively eliminated Stokes-shifted autofluorescence from plasma samples.
• The technique enabled clear detection of fluorescence bursts from UCNPs.
• A homogeneous sandwich immunoassay for TSH was successfully performed in both buffer and plasma using UCCS.
• Demonstrated the feasibility of UCCS for sensitive and specific biomarker detection in complex biological fluids.

Conclusions:

• Upconversion Cross-Correlation Spectroscopy (UCCS) provides a robust solution to overcome autofluorescence limitations in biological analyses.
• UCCS facilitates accurate measurements in complex matrices such as plasma, expanding the applicability of correlation spectroscopy.
• This method holds significant potential for advancing sensitive immunoassays and other diagnostic applications in clinical settings.