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Novel anti-Stokes lanthanide-doped nanoparticles and multicolor FRET mechanisms for single-molecule DNA sequencing (LantaSEQ)

Principal investigator: prof. dr hab. Artur Bednarkiewicz 
Foreign co-principal investigator: Assoc. Prof. Hans Gorris Ph.D

Single-molecule DNA sequencing is highly desirable in molecular diagnostics due to:

  • Simplified DNA template preparation,

  • Higher throughput and speed,

  • Longer reading, leading to reduced costs compared to conventional sequencing techniques.

However, current single-molecule DNA sequencing methods have several limitations that can be overcome by exploiting the unique photophysical properties of new lanthanide-doped luminescent nanomaterials (LnNPs):

  • LnNPs efficiently emit visible light under NIR excitation with large anti-Stokes shifts >300 nm. Under these conditions, no other sample components are excited, and background signals from autofluorescence and light scattering are eliminated.

  • LnNPs are photostable and, unlike quantum dots, do not exhibit blinking. Consequently, these new nanoparticles provide a stable donor signal for FRET, which is essential for continuous readout of long DNA sequences.

  • LnNPs emit multiple narrow emission bands under single-wavelength excitation, allowing a single luminescent nanoparticle to serve as a FRET donor for up to four different acceptor dyes required to identify individual dNTPs.

Single-molecule sequencing using lanthanide-doped nanoparticles is a promising approach, but it has never been realized before. To achieve this, it is necessary to:

  • Optimize optical readout instruments for detecting single LnNPs and for wide-field microscopic imaging.

  • Synthesize and study new, specially designed LnNPs by optimizing dopant architecture and core–shell composition to maximize brightness and sensitivity to acceptor molecules.

  • Optimize polymerase surface conjugation to minimize the distance between UCNPs and polymerase.

  • Apply the newly developed materials, instruments, and methods to sequence test DNA molecules.