Abstract
An ultraefficient cap-exchange protocol (UCEP) that can convert hydrophobic quantum dots (QDs) into stable, biocompatible, and aggregation-free water-dispersed ones at a ligand:QD molar ratio (LQMR) as low as 500, some 20–200-fold less than most literature methods, has been developed. The UCEP works conveniently with air-stable lipoic acid (LA)-based ligands by exploiting tris(2-carboxylethyl phosphine)-based rapid in situ reduction. The resulting QDs are compact (hydrodynamic radius, Rh, < 4.5 nm) and bright (retaining > 90% of original fluorescence), resist nonspecific adsorption of proteins, and display good stability in biological buffers even with high salt content (e.g., 2 M NaCl). These advantageous properties make them well suited for cellular imaging and ratiometric biosensing applications. The QDs prepared by UCEP using dihydrolipoic acid (DHLA)-zwitterion ligand can be readily conjugated with octa-histidine (His8)-tagged antibody mimetic proteins (known as Affimers). These QDs allow rapid, ratiometric detection of the Affimer target protein down to 10 pM via a QD-sensitized Förster resonance energy transfer (FRET) readout signal. Moreover, compact biotinylated QDs can be readily prepared by UCEP in a facile, one-step process. The resulting QDs have been further employed for ratiometric detection of protein, exemplified by neutravidin, down to 5 pM, as well as for fluorescence imaging of target cancer cells.
Highlights
Over the past two decades, quantum dots (QDs) have been of significant research focus due to their unique, size-dependent, stable, and bright fluorescence, making them powerful probes for a wide range of applications such as energy, materials, biology, and medicine.[1−11] Their broad absorption and stable, narrow symmetric emission are well suited for multiplexed sensing, biodiagnostics, bioimaging, immunoassay, cell tracking, and trafficking studies.[3−6,9,12−20] In this regard, a robust, compact, and biocompatible QD structure is of paramount importance
It remained a laborious and delicate process because the dihydrolipoic acid (DHLA)-based ligands had to be freshly prepared, purified by column chromatography, and used in the same day to ensure a successful cap exchange. This is because the DHLA-based ligands are air sensitive and susceptible to oxidization to their lipoic acid (LA) forms, which results in loss of QD binding affinity.[46]
We show that ultraefficient cap-exchange protocol (UCEP) can completely transform the hydrophobic QDs into stable, aggregation-free water dispersions at a ligand:QD molar ratio (LQMR) as low as 500, ∼20−200-fold lower than most current literature protocols (Table 1)
Summary
Over the past two decades, quantum dots (QDs) have been of significant research focus due to their unique, size-dependent, stable, and bright fluorescence, making them powerful probes for a wide range of applications such as energy, materials, biology, and medicine.[1−11] Their broad absorption and stable, narrow symmetric emission are well suited for multiplexed sensing, biodiagnostics, bioimaging, immunoassay, cell tracking, and trafficking studies.[3−6,9,12−20] In this regard, a robust, compact, and biocompatible QD structure is of paramount importance. It remained a laborious and delicate process because the DHLA-based ligands had to be freshly prepared, purified by column chromatography, and used in the same day to ensure a successful cap exchange This is because the DHLA-based ligands are air sensitive and susceptible to oxidization to their LA forms, which results in loss of QD binding affinity.[46] By contrast, the photoligation method developed by the Mattoussi group can work directly with the air-stable LA form of ligands.[46−49] the requirement for a large excess of ligands The resulting QDs are compact (Rh < 4.5 nm), retain > 90% of their original fluorescence, and resist nonspecific adsorption, making them powerful fluorescence probes for FRET-based ratiometric sensing and cancer cell imaging
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