Biology subjects

Alexaki, K.

Publications and source records attributed to Alexaki, K..

3 recordsLinked to original sources

bioRxiv2026

Mechanistic Insights into Magnesium Pyrophosphate Formation in the Presence of Gold Nanoclusters Enable Genetic Analysis via Co-Aggregation-Induced Fluorescence Enhancement

The formation of magnesium pyrophosphate (Mg2P2O7) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg2P2O7 serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg2P2O7 remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg2P2O7-driven co-aggregation mechanism involving glutathione-capped gold nanoclusters (GSH-AuNCs) that induces fluorescence enhancement, enabling real-time crystal formation monitoring. The mechanism was first investigated in simplified mixtures containing pyrophosphate (P2O74-) and magnesium (Mg2+) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg2P2O7 co-aggregation can be correlated with crystal formation/growth/solubilization and solution turbidity, while distinct kinetic patterns can be indicative of the crystal size at the end of the reaction. As a next level of complexity, we examined the effects of common components in an enzymatic amplification reaction, i.e., dithiothreitol (DTT), ammonium sulfate ((NH4)2SO4), deoxynucleotides (dNTPs) and Bst polymerase, on Mg2P2O7 formation through real-time GSH-AuNCs fluorescence variations. Guided by the above results, we studied and selected the experimental parameters for the design of an optimized qualitative (end-point) or quantitative (real-time) genetic test. Finally, the loop-mediated isothermal amplification (LAMP) was used as a platform to demonstrate the quantification of Influenza A RNA within the range of 102-108 copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of <25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg2P2O7 formation in other biotechnological processes, including in vitro transcription and Mg2P2O7-bioorganic composites synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/744482v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@42206dorg.highwire.dtl.DTLVardef@15edc71org.highwire.dtl.DTLVardef@dab2b5org.highwire.dtl.DTLVardef@aa535_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Single cell RNA sequence analysis of human bone marrow samples reveals new targets for isolation of skeletal stem cells using DNA-coated gold nanoparticles

There is a wealth of data indicating human bone marrow derived stromal cells (HBMSCs) contain the skeletal stem cell (SSC) with the potential to differentiate along the stromal osteogenic, adipogenic and chondrogenic lineages. However, despite these advances, current methods to isolate skeletal stem cells (SSCs) from human tissues have proved challenging as no single specific marker has been identified limiting understanding of SSC fate, immunophenotype and the widespread clinical application of these cells. While a number of cell surface markers can enrich for SSCs, none of the proposed markers, alone, provide a platform to isolate single cells with the ability to form bone, cartilage, and adipose tissue in humans. The current study details the application of oligonucleotide-coated nanoparticles, spherical nucleic acids (SNAs), to rapidly isolate human cells using mRNAs signatures detected in SSCs in real time, to identify stem and progenitor skeletal populations using single cell RNA sequencing. Based on scRNA-seq of samples from 11 patients, this method was able to identify novel targets for SSC enrichment, which were assessed in a total of 80 patients. This methodology was able to isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with a capacity for tri-lineage differentiation in vitro. The current approach provides new targets and a platform to advance SSC isolation, enrichment with significant therapeutic impact therein.

cell biology↗

bioRxiv2019

Optimising DNA-Coated Gold Nanoparticles to Enrich Skeletal Stem Cells from Human Bone Marrow Samples

Human bone marrow (BM) derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic and chondrogenic lineages enabling their application to clinical therapies. However, current methods, to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers, alone, can isolate a homogenous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications. TABLE OF CONTENTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/882563v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@48c5acorg.highwire.dtl.DTLVardef@1a9d096org.highwire.dtl.DTLVardef@1bd4da2org.highwire.dtl.DTLVardef@133dd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗