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UV–Vis Spectroscopy of Conjugated Systems01:32

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Organic compounds with conjugated double bonds show strong absorption features in the UV–visible region of the electromagnetic spectrum attributed to π → π* electronic excitations. Generally, a UV–vis absorption spectrum is recorded as a plot of absorbance vs wavelength. The wavelength of maximum absorbance, which manifests as a peak in the absorption spectrum, is denoted as λmax.
One of the factors influencing λmax is the extent of conjugation in...
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Heterogeneity of Upconversion Bioconjugates Revealed by Single Nanoparticle Spectroscopy.

Farrel Rafaelino Separgo1, Yangjian Cai1, Yitong Zhao1

  • 1Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, New South Wales 2007, Australia.

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|November 25, 2025
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Summary
This summary is machine-generated.

Researchers developed a new microscopy method to analyze lanthanide-doped upconverting nanoparticles (UCNPs) conjugated to antibodies. This technique reveals significant variability in antibody attachment, crucial for improving UCNP-based biomedical tools.

Keywords:
antibody quantificationconjugatesheterogeneitysingle-particle analysisupconverting nanoparticles (UCNPs)

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Area of Science:

  • Nanotechnology
  • Bioconjugation
  • Microscopy

Background:

  • Lanthanide-doped upconverting nanoparticles (UCNPs) are vital for bioimaging and biosensing.
  • Their functionality depends on conjugation with biomolecules like antibodies for specific targeting.
  • Assessing conjugation consistency at the single-particle level is challenging but critical.

Purpose of the Study:

  • To develop and apply a correlative microscopy approach for single-particle analysis of UCNP-antibody conjugates.
  • To quantify antibody loading and heterogeneity on UCNPs.
  • To understand factors influencing conjugate formation and performance.

Main Methods:

  • Correlative microscopy combining upconversion and downshifting imaging.
  • Photobleaching analysis to quantify antibody (Alexa-647 labeled anti-spike) loading.
  • Distinguishing single UCNPs from clusters using upconversion emission.

Main Results:

  • Demonstrated substantial heterogeneity in UCNP-antibody conjugates, including unconjugated particles, single conjugates, and clusters.
  • Quantified varied antibody numbers per UCNP, averaging up to six.
  • Identified antibody structural changes as a factor promoting cluster formation.

Conclusions:

  • The single-particle analysis strategy enables direct quantification of nanoconjugate heterogeneity.
  • Provides critical insights for optimizing the design and performance of UCNP-based biomedical tools.
  • Highlights the importance of understanding conjugation variability for reliable nanomedicine applications.