Phase-Change Partitions for Thermal Automation of Multistep Reactions.

Medical diagnostics and basic research in low-resource settings require automated reactions to be controlled in a simple, portable manner. Here, we present a novel platform that enables simple automation of multistep reactions to facilitate robust, hands-free assay operation without complex microfluidics or paperfluidics. We separate reagent zones in a conventional PCR tube via solid layers of purified higher alkanes. Reagents can be mixed on demand by simply raising the temperature above the melting point of the alkane partition that separates the two zones. We partitioned various reagents to enable hands-free thermally automated isothermal nucleic acid amplification, heavy metal ion detection, and ß-lactamase detection with tandem antibiotic specificity characterization. We anticipate that this phase-change partition platform will find broad application in clinical diagnostics at the point-of-care and in low-resource settings.

Peroxidyme-Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter.

Peroxidyme Amplified Radical Chain Reaction (PARCR), a novel enzyme-free system that achieves exponential amplification of a visible signal, is presented. Typical enzyme-free amplification systems that produce a visible readout suffer from long reaction times, low sensitivity, and narrow dynamic range. PARCR employs photocatalyzed nonlinear signal generation, enabling unprecedented one-pot, naked-eye detection of a catalytic reporter from 1 µm down to 100 pm. In this reaction, hemin-binding peroxidase-mimicking DNAzymes ("peroxidymes") mediate the NADH-driven oxidation of a colorless, nonfluorescent phenoxazine dye (Amplex Red) to a brightly colored, strongly fluorescent product (resorufin); illumination with green light initiates multiple radical-forming positive-feedback loops, rapidly producing visible levels of resorufin. Collectively, these results demonstrate the potential of PARCR as an easy-to-use readout for a range of detection schemes, including aptamer labels, hybridization assays, and nucleic acid amplification.

Transfer Learning Bayesian Optimization to Design Competitor DNA Molecules for Use in Diagnostic Assays.

With the rise in engineered biomolecular devices, there is an increased need for tailor-made biological sequences. Often, many similar biological sequences need to be made for a specific application meaning numerous, sometimes prohibitively expensive, lab experiments are necessary for their optimization. This paper presents a transfer learning design of experiments workflow to make this development feasible. By combining a transfer learning surrogate model with Bayesian optimization, we show how the total number of experiments can be reduced by sharing information between optimization tasks. We demonstrate the reduction in the number of experiments using data from the development of DNA competitors for use in an amplification-based diagnostic assay. We use cross-validation to compare the predictive accuracy of different transfer learning models, and then compare the performance of the models for both single objective and penalized optimization tasks.

Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging.

Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery.

PURPOSE: To report a case of diffuse lamellar keratitis (DLK) after corneal collagen cross-linking in an eye with a remote history of laser in situ keratomileusis (LASIK) surgery. METHODS: This is a case report and literature review. RESULTS: This report describes the development of unilateral stage IV DLK in a patient who underwent bilateral corneal cross-linking for corneal ectasia 18 years after LASIK surgery. The patient was treated with high-dose topical steroids that were tapered over 1 month and multiple flap lifts. The ultimate best-corrected visual outcome was 20/60. CONCLUSIONS: DLK is a potential sight-threatening complication of refractive surgery that can occur at any time in the postoperative period, even years after the procedure. Undergoing a subsequent corneal procedure that may disrupt or promote inflammation within the surgical flap-stromal interface, such as corneal collagen cross-linking, is a recognized risk factor for the development of DLK. This case suggests that patients with any history of LASIK surgery undergoing corneal cross-linking or other lamellar corneal surgeries may benefit from closer follow-up (eg, daily) than patients with no history of LASIK.

Kinetics of RNA and RNA:DNA Hybrid Strand Displacement.

In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

Multistage Chemical Heating for Instrument-Free Biosensing.

Improving the portability of diagnostic medicine is crucial for alleviating global access-to-care deficiencies. This requires not only designing devices that are small and lightweight, but also autonomous and independent of electricity. Here, we present a strategy for conducting automated multistep diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for rolling circle amplification of nucleic acids are separated by meltable phase-change partitions, thus replacing precise manual reagent additions with automated partition melting. To actuate these barriers and individually initiate the various steps of the reaction, field ration heaters exothermically generate heat in a thermos, whereas fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multistage temperature profiles extend the capability of instrument-free diagnostic devices and improve the portability of reaction automation systems built around phase-change partitions.

Fabrication of rigid microstructures with thiol-ene-based soft lithography for continuous-flow cell lysis.

In this work, we introduce a method for the soft-lithography-based fabrication of rigid microstructures and a new, simple bonding technique for use as a continuous-flow cell lysis device. While on-chip cell lysis techniques have been reported previously, these techniques generally require a long on-chip residence time, and thus cannot be performed in a rapid, continuous-flow manner. Microstructured microfluidic devices can perform mechanical lysis of cells, enabling continuous-flow lysis; however, rigid silicon-based devices require complex and expensive fabrication of each device, while polydimethylsiloxane (PMDS), the most common material used for soft lithography fabrication, is not rigid and expands under the pressures required, resulting in poor lysis performance. Here, we demonstrate the fabrication of microfluidic microstructures from off-stoichiometry thiol-ene (OSTE) polymer using soft-lithography replica molding combined with a post-assembly cure for easy bonding. With finite element simulations, we show that the rigid microstructures generate an energy dissipation rate of nearly 10(7), which is sufficient for continuous-flow cell lysis. Correspondingly, with the OSTE device we achieve lysis of highly deformable MDA-MB-231 breast cancer cells at a rate of 85%, while a comparable PDMS device leads to a lysis rate of only 40%.

Visualization of recombinant DNA and protein complexes using atomic force microscopy.

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation, such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase and a repair enzyme, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes) and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.