Facile Preconcentration of Cell-Free DNA in Human Plasma by Ion-Specific Poly Ionic Sorbents Featuring an Anion Exchange Mechanism.

The expanding horizon of diagnostic and therapeutic applications involving nucleic acids (NA) requires novel tools for purification, including minimal sample preparation. In this work, thin-film microextraction devices featuring five poly ionic sorbents were examined as anion exchange extraction phases for the rapid purification of NAs. Each sorbent is composed of a nonionic cross-linker and a methacrylate monomer containing a core tetra-alkyl ammonium moiety with an alkyl, anionic, or cationic residue. Extraction devices were produced through the application of the prepolymer sorbent mixture onto a functionalized nitinol metal support followed by photoinduced free-radical polymerization. The miniaturized extraction devices (10 mm × 3.5 mm) were directly immersed into aqueous samples to isolate NAs via electrostatic interactions with the polycation. The ammonium methacrylate (AMA) monomer containing a propyl trimethylammonium group (AMA-C3N(CH3)3) exhibited the highest affinity for DNA, with 80 ± 10% of DNA being isolated. Recovery of DNA from the sorbents required the introduction of ions in an aqueous solution to exchange the anionic biopolymer from the polycationic moiety. An investigation of three anion species revealed that the AMA-C3N(CH3)3 sorbent showed the highest recoveries, with the perchlorate anion producing a preconcentration factor of 4.36 ± 0.86 while requiring only 250 mM NaClO4. A directly compatible quantitative polymerase chain reaction assay was developed to quantify the recovery of spiked DNA with lengths of 830, 204, and 98 base pairs in heat-treated human plasma. The AMA-C3N(CH3)3 sorbent was uninhibited by the complex human plasma matrix and enabled high preconcentration factors for the spiked DNA at a biologically relevant concentration of 10 pg/mL. While Qiagen's circulating cell-free DNA MinElute extraction kit enabled higher preconcentration of all analytes, the methodology described in this work requires fewer steps, less user intervention, and minimal equipment requirements to isolate DNA, making it more amenable for high-throughput and low resource applications.

Versatile dual-channel loop-mediated isothermal amplification assay featuring smartphone imaging enables determination of fecal indicator bacteria in environmental waters.

Rapid diagnostic assays are often a critical tool for monitoring water quality in developing and developed countries. Conventional testing requires 24-48 h for incubation, resulting in delayed remediation and increasing the likelihood of negative outcomes. In this study, we report a workflow for detection of E. coli, a common indicator of fecal contamination. Following large volume filtration, E. coli is then solubilized enabling the facile isolation and recovery of genetic material by a thin film microextraction (TFME) device featuring a polymeric ionic liquid (PIL) sorbent. Rapid recovery of pure nucleic acids is achieved using a PIL sorbent with high affinity for DNA to significantly increase mass transfer and facilitate adsorption and desorption of DNA. Downstream detection is performed by a versatile, dual channel loop mediated isothermal amplification (LAMP) assay featuring a colorimetric dye and a sequence-specific molecular beacon. A portable LAMP companion box enables consistent isothermal heating and endpoint smartphone imaging while being powered by a single 12-V battery. Programmable LEDs are switched from white or blue light to facilitate the independent imaging of the colorimetric dye or fluorometric probe following amplification. The methodology positively identified E. coli in environmental samples spiked to concentrations of 6600 colony forming units (CFU) per milliliter and 660 CFU/mL with 100% and 22% positivity, respectively.

Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection.

Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.

Evaluating commercial thermoplastic materials in fused deposition modeling 3D printing for their compatibility with DNA storage and analysis by quantitative polymerase chain reaction.

Nucleic acids are ubiquitous in biological samples and can be sensitively detected using nucleic acid amplification assays. To achieve highly accurate and reliable results, nucleic acid isolation and purification is often required and can limit the accessibility of these assays. Encapsulation of these workflows onto a single device may be achieved through fabrication methodologies featuring commercial three-dimensional (3D) printers. This study aims to characterize fused deposition modeling (FDM) filaments based on their compatibility with nucleic acid storage using quantitative polymerase chain reaction (qPCR). To study the adsorption of nucleic acids, storage vessels were fabricated using six common thermoplastics including: polylactic acid (PLA), nylon, acrylonitrile butadiene styrene (ABS), co-polyester (CPE), polycarbonate (PC), and polypropylene (PP). DNA adsorption of a short 98 base pair and a longer 830 base pair fragment to the walls of the vessel was shown to vary significantly among the polymer materials as well as the color varieties of the same polymer. PLA storage vessels were found to adsorb the least amount of the 98 base pair DNA after 12 hours of storage in 2.5 M NaCl TE buffer whereas the ABS and PC vessels adsorbed up to 97.2 ± 0.2% and 97.5 ± 0.2%. DNA adsorption could be reduced by decreasing the layer height of the 3D printed object, thereby increasing the functionality of the ABS storage vessel. Nylon was found to desorb qPCR inhibiting components into the stored solution which led to erroneous DNA quantification data from qPCR analysis.

Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays.

Nucleic acid analysis has been at the forefront of the COVID-19 global health crisis where millions of diagnostic tests have been used to determine disease status as well as sequencing techniques that monitor the evolving genome of SARS-CoV-2. In this study, we report the development of a sample preparation method that decreases the time required for DNA isolation while significantly increasing the sensitivity of downstream analysis. Functionalized planar supports are modified with a polymeric ionic liquid sorbent coating to form thin film microextraction (TFME) devices. The extraction devices are shown to have a high affinity for DNA while also exhibiting high reproducibility and reusability. Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times while achieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapid desorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediated isothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystal violet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oral fluid samples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7% obtained by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.

Sequence-Specific Detection of ORF1a, BRAF, and ompW DNA Sequences with Loop Mediated Isothermal Amplification on Lateral Flow Immunoassay Strips Enabled by Molecular Beacons.

Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.

Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay.

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.