Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >10(5)

High performance methylated DNA markers for detection of colon adenocarcinoma

BACKGROUND: Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA. METHODS: Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy. RESULTS: In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue. CONCLUSIONS: We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13148-021-01206-2

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1–DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC(50) of DOX on the dissociation of Sp1–DNA complex is estimated to be 0.55 μM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques

Electromagnetic Wave Theory and Applications

Contains table of contents for Section 3, reports on three research projects and a list of publications.California Institute of Technology/Jet Propulsion Laboratory Contract 959548National Aeronautics and Space Administration Grant NAGW-1617National Aeronautics and Space Administration Grant Contract 958461U.S. Navy - Office of Naval Research Grant N00014-92-J-1616U.S. Navy - Office of Naval Research Grant N00014-92-J-4098Digital Equipment Corporation AGMT DTD 11/16/93Joint Services Electronics Program Contract DAAL03-92-C-0001Joint Services Electronics Program Grant DAAH04-95-1-0038MIT Lincoln Laboratory P.O. No. BX-5424U.S. Navy - Office of Naval Research Grant N00014-90-J-1002U.S. Navy - Office of Naval Research Grant N00014-89-J-1019DEMACO Agreement 11/15/93Federal Aviation Administration Grant 94-G-007U.S. Army Cold Regions Research and Engineering Laboratory Contract DACA89-93-K-000

Micro and Nanotechnologies Enhanced Biomolecular Sensing

This editorial summarizes some of the recent advances of micro and nanotechnology-based tools and devices for biomolecular detection. These include the incorporation of nanomaterials into a sensor surface or directly interfacing with molecular probes to enhance target detection via more rapid and sensitive responses, and the use of self-assembled organic/inorganic nanocomposites that inhibit exceptional spectroscopic properties to enable facile homogenous assays with efficient binding kinetics. Discussions also include some insight into microfluidic principles behind the development of an integrated sample preparation and biosensor platform toward a miniaturized and fully functional system for point of care applications

Rapid generation of chemical combinations on a magnetic digital microfluidic array

Combinatorial screening is frequently used to identify chemicals with synergistic effects by measuring the response of biological entities exposed to various chemical-dose combinations. Conventional microwell-based combinatorial screening is resource-demanding, and the closed microfluidics-based screening requires sophisticated fluidic control systems. In this work, we present a novel combinatorial screening platform based on the surface energy trap (SET)-assisted magnetic digital microfluidics. This platform, known as FlipDrop, rapidly generates chemical combinations by coupling two droplet arrays with orthogonal chemical concentration gradients with a simple flip. We have illustrated the working principle of FlipDrop by generating combinations of quantum dots. We have also successfully demonstrated the screening of quantum dot fluorescence resonance energy transfer (QD-FRET) on the FlipDrop platform by measuring the FRET response. This report demonstrates that FlipDrop is capable of rapidly generating chemical combinations with unprecedented ease for combinatorial screening.MOE (Min. of Education, S’pore)Published versio

PAPER www.rsc.org/analyst | The Analyst

Comparative quantification of nucleic acids using single-molecule detection and molecular beacon

Enhancing Throughput of Combinatorial Droplet Devices via Droplet Bifurcation, Parallelized Droplet Fusion, and Parallelized Detection

Combinatorial droplet microfluidic devices with programmable microfluidic valves have recently emerged as a viable approach for performing multiplexed experiments in microfluidic droplets. However, the serial operation in these devices restricts their throughput. To address this limitation, we present a parallelized combinatorial droplet device that enhances device throughput via droplet bifurcation, parallelized droplet fusion, and parallelized droplet detection. In this device, sample droplets split evenly at bifurcating Y-junctions before multiple independent reagent droplets are injected directly into the split sample droplets for robust droplet fusion. Finally, the fused sample and reagent droplets can be imaged in parallel via microscopy. The combination of these approaches enabled us to improve the throughput over traditional, serially-operated combinatorial droplet devices by 16-fold—with ready potential for further enhancement. Given its current performance and prospect for future improvements, we believe the parallelized combinatorial droplet device has the potential to meet the demand as a flexible and cost-effective tool that can perform high throughput screening applications