16 research outputs found
Single Molecule Detection : Microfluidic Automation and Digital Quantification
Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity. This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics
Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
Label-free monitoring of protein-DNA interactions using fluorescent silver nanoclusters
Aptamers are short ssDNA or RNA oligonucleotides that bind targets with high affinity and specificity by folding into defined tertiary structures. Thanks to these properties aptamers represent attractive bio-receptors.[1][2] Traditionally new aptamers are obtained through SELEX, an iterative process in which sequences are selected against target molecules.[3] Generally, aptamer-target interactions are detected in indirect ways relying on labeling of either target or aptamer. However, labeling is not always desirable and label-free detection would be valuable for sensitive detection of binding events. This work explores the use of fluorescent silver nanoclusters (AgNCs) to develop an innovative system for monitoring aptamer-protein interactions.
AgNCs are complexes between few Ag atoms and a specific DNA sequence template to stabilize the clusters. In most cases, AgNCs are synthesized either at the 3’ or 5’ end of the template. Our results show that AgNCs can successfully be generated from a template embedded in the middle of a hybridization probe, used for the isothermal amplification of aptamers, called rolling circle amplification (RCA). RCA uses circular oligonucleotide probes to generate long, ssDNA molecules containing periodic repeats of the circular probe.[4][5] Previous works show that overexpression of aptamers by RCA increases target binding efficiency compared to monovalent aptamers.[6] The RCA concatemer combines both the aptamer and the fluorescent AgNC template. Subsequently synthetized AgNCs exhibit strong, robust and tunable fluorescence, eliminating the need for labeling.[7] Importantly, it has been shown that aptamer-AgNCs retain the same specificity and affinity for the cognate protein and that target binding results in a drastic decrease of nanocluster fluorescence.[8]
This work explores the integration of an AgNC-aptamer sequence into a circular probe to generate intrinsically fluorescent aptamer concatemers with improved binding efficiency. Possible applications are monitoring target binding during SELEX and label-free ultrasensitive detection of proteins.status: publishe
Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules
Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as similar to 1 pg (similar to 300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.De 2 första författarna delar förstaförfattarskapet.</p
Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules
Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy
Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies
Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease
Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-μm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases