Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32-element array with ~80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn-key, high-yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low-cost, parallelizable, and automatable, allowing high-throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.

Target DNA fragments at 10 fM concentration (approximately 6 × 105 molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences.

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

Artificial lipid bilayers are well established for use in scientific studies of reconstituted ion channels and as a platform to host engineered pore proteins for sensing, including DNA sequencing. Droplet bilayers have recently been shown to be especially compatible with technological applications, with recently demonstrated automation, parallelization, and ion channel drug potency measurements. To expand the range of potential applications, we have been working to simplify droplet bilayer formation and ion channel measurement. We have developed a chip for droplet bilayer formation and measurement that only requires fluid dispensation. Using an array design of this chip, 32 bilayers were simultaneously formed with no operator feedback at a yield of approximately 80%. Cycling this process resulted in the formation and measurement of 96/120 bilayers in 80 minutes, a rate which could greatly increase with automation and greater parallelization. We also used these arrays to measure the a-hemolysin and VDAC channels.

Հոդվածում ներկայացված է Դվին հնավայրի մ. թ. ա. III հազարամյակից սկսած մինչև մ. թ. IV դարն ընդգրկող մշակութային շերտերում ամրոց-սրբավայր զույգի ավանդապահ տևականության բնութագիրը: Պատմամշակութային ժառանգականոթյան հենքի վրա նորովի է վերանայվում հնագիտական և գրավոր սկզբնաղբյուրների առկա տեղեկատվությունը: Նման մոտեցումը հնարավորություն է ընձե¬ռում ուրվագծելու ժառանգված իրողությունների զարգացման բնականոն ընթացքի ուղին Դվին բազմաշերտ հնավայրում: