Grayscale lithography can be used to create a three-dimensional topography in the substrate. This method can be utilized in the fabrication of microfluidic chips, for lab-on-chip (LoC) applications. Here, a grayscale lithography process is shown that creates gradient inlet structures for nanochannels. Nanochannels are a tool for optical DNA analysis, when the molecules are linearized inside of the channels. A pixelated grayscale mask was designed for the process with a minimal structure size of 300 nm, which still allowed the creation of structures with features in the size of $1 \mu ~\mathrm{m}$ without the need for a reduction system. An etch recipe was created that etches the substrate with the same etch rate as the photoresist to transfer the topology into the substrate.

Next-generation sequencing (NGS) is becoming more relevant for medical diagnostics, especially for using cell-free DNA to monitor response to therapy in cancer management, as high sensitivity of NGS enables detection of rare events. Sequencing Library preparation is a time-consuming and complex process, and large-scale liquid handlers are often used for automation. However, for smaller labs and low-to-medium throughput samples, these liquid handlers are expensive and need experts for handling. This work presents a proof-of-concept for library preparation on a commercially available and open lab-on-a-chip platform, which provides an alternative automation for low-to-medium throughput requirements. It covers common library preparation steps optimized to a microfluidic environment that include customizable PCR for target enrichment, end-repair, adapter ligation, nucleic acid purification via magnetic beads, and an integrated quantification step. The functionality of the cartridge is demonstrated with reference cfDNA containing different allelic frequencies of seven known mutations. Processing the samples in the cartridge reveals highly comparable results to manual processing (Pearson r = 0.94) based on amplicon sequencing. Summarized, the proposed automated lab-on-a-chip workflow for customizable library preparation could further pave the way for NGS to evolve from a technology used for research purposes to one that is applied in routine cancer management.

We investigate the transport of electrons in a ferrocene aqueous solution nanoconfined between two Pt electro-des using density functional theory and a nonequilibrium Green function method. The system consists of three characteristic phases: metal electrodes, the electrode-solution interface, and the nanoconfined solution phase. By performing the geometry optimization of such systems, it is found that the molecular configuration of water molecules at the Pt surface is adjusted due to the Pt-water interaction, and the charges at the Pt surface are redistributed. Next, by applying the external bias potential via the effective screening medium method during ab initio molecular dynamics simulations, it is revealed that water molecules are packed at the Pt-water interfacial region, and the electrostatic potential profile in the water phase is significantly changed due to aligned water layers. From the analysis of the electron transport characteristics, it is discovered that the ferrocene molecule generates a strong transmission function near the Fermi level and high electron density of states spatially connecting both electrodes through the water phase. These features explain the drastic enhancement in electrical current-voltage characteristics. Therefore, it is concluded that ferrocene enhances electron transport through nanoconfined solutions, indicating that it can be used as a signaling molecule in nanoscale systems for electrochemical sensor applications.

An electrochemical platform for generating and controlling a localized pH microenvironment on demand is proposed by employing a closed-loop control algorithm based on an iridium oxide pH sensor input. We use a combination of solution-borne quinones and galvanostatic excitation on a prepatterned indium tin oxide (ITO) working electrode to modulate pH within a very well confined, small volume of solution close to the electrode surface. We demonstrate that the rate of pH change can be controlled at up to 2 pH s-1 with an excellent repeatability (±0.004). The desired pH microenvironment can be stably maintained for longer than 2 h within ±0.0012 pH. As a high-impact application of the platform technology, we propose a single-step immunoassay and demonstrate its utility in measuring C-reactive protein (CRP), a critical inflammatory marker in various conditions such as myocardial infarction and even SARS-Cov-2. Utilizing pH modulation technology along with pH-sensitive fluorescence dye simplifies the immunoassay process into a single-step, where a mixture of all of the reagents is incubated only for 1 h without any washing steps or the need to change solution. This simplified immunoassay process minimizes the hands-on time of the end-user and thus decreases technician-driven errors. Moreover, the absence of complicated liquid-handling hardware makes it more suitable and attractive for an ultracompact platform to ultimately be used in a point-of-care diagnostic assay.

Al2O3 is commonly used in modern electronic devices because of its good mechanical properties and excellent electrical insulating property. Although fundamental understanding of the electron transport in Al2O3 is essential for its use in electronic device applications, a thorough investigation for the electron-transport mechanism has not been conducted on the structures of Al2O3, especially in nanometer-scale electronic device settings. In this work, electron transport via Al2O3 for two crystallographic facets, (100) and (012), in a metal-insulator-metal junction configuration is investigated using a density functional theory-based nonequilibrium Green function method. First, it is confirmed that the transmission function, T(E), decreases as a function of energy in (E - E F) < 0 regime, which is an intuitively expected trend. On the other hand, in the (E - E F) > 0 regime, Al2O3(100) and Al2O3(012) show their own characteristic behaviors of T(E), presenting that major peaks are shifted toward lower energy levels under a finite bias voltage. Second, the overall conductance decay rates under zero bias are similar regardless of the crystallographic orientation, so that the contact interface seemingly has only a minor contribution to the overall conductance. A noteworthy feature at the finite bias condition is that the electrical current drastically increases as a function of bias potential (>0.7 V) in Al2O3(012)-based junction compared with the Al2O3(100) counterpart. It is elucidated that such a difference is due to the well-developed eigenchannels for electron transport in the Al2O3(012)-based junction. Therefore, it is evidently demonstrated that at finite bias condition, the contact interface plays a key role in determining insulating properties of Al2O3-Pt junctions.

Abstract Background. Mutation detection through genetic testing is playing an increasingly important role in personalized precision medicine in cancer. However, current tests identifying driver mutations as therapeutic targets are based on detection of common mutations in cancer genes. These tests are not patient specific and do not address intra-tumor heterogeneity. Ubiquitous intra-tumor genetic heterogeneity is a mechanism of drug resistance and cancer recurrence. Methods. Approximately 16-24 microsamples are acquired to represent the entire cancer cell population for every ovarian tumor. Each microsample consists of a few cells within a clone and is selected to substitute for a single cell and overcome the large allele dropout rate commonly seen in single genome amplification and sequencing. TEAPOT (Tumor Evolution Assay for Personalized Oncology Therapy) algorithm has been developed to reconstruct a tumor's evolutionary history through integration of whole exome sequencing data from the bulk primary tumor and 16-24 microsamples taken from the bulk tumor. The evolutionary history for an individual tumor is expressed as a rooted and binary tumor developmental tree representing the mitotic process starting from an ancestral cancer cell. Individual mutations are assigned to the cells where they originally occur. The offspring size carrying a mutation was estimated based on tumor purity, variant allele frequency and the variant's copy number. Results. TEAPOT algorithm builds a tumor's evolutionary history with the following features: 1) a tumor's evolutionary history is unique for each ovarian cancer patient; 2) the size of a tree is proportional to the number of microsamples selected; 3) 16-24 microsamples builds a tree with 5 or more generations; 4) TEAPOT detects a driver mutation's occurrence at a specific developmental stage such as 1-cell, 2-cell, 4-cell, etc; 5) The size of offspring carrying a mutation thus the intra-tumor prevalence of the mutation can be estimated; 6) multiple driver mutations can be located separately in different clones. Therefore, TEAPOT provides a quantitative description of intra-tumor genetic heterogeneity and identifies sub-clonal driver mutations in a tumor. Conclusion. TEAPOT reconstructs a tumor's developmental process thus providing a patient-specific evolutionary history. Quantitation of intra-tumor prevalence of driver mutations may inform selection of an effective targeted agent and may provide rationale for cocktail treatment targeting multiple driver mutations simultaneously. TEAPOT can be also used for other solid and liquid cancers. A driver mutation's role in a patient may be functionally defined and quantitated based upon the growth advantage (fitness) it confers on its host cells in the reconstructed tumor evolutionary history. Citation Format: Jianshu Zhang, Helaman Escobar, Harshmi Shah, Mickey Miller, Yang Wei, Kristen Schneider, Michelle Knirr, Kenny Day, Christopher Johnson, Baoli Yang, Eric Devor, Kristina Thiel, Lincoln Nadauld, Kimberly Leslie, Donghai Dai. Development of TEAPOT algorithm to reconstruct individual ovarian tumors' evolutionary history based upon bulk and single cell whole exome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 219.

Solution pH is a powerful tool for regulating many kinds of chemical activity, but is generally treated as a static property defined by a pre-selected buffer. Introducing dynamic control of pH in space, time, and magnitude can enable richer and more efficient chemistries, but is not feasible with traditional methods of titration or buffer exchange. Recent reports have featured electrochemical strategies for modifying bulk pH in constrained volumes, but only demonstrate switching between two preset values and omit spatial control entirely. Here, we use a combination of solution-borne quinones and galvanostatic excitation to enable quantitative control of pH environments that are highly localized to an electrode surface. We demonstrate highly reproducible acidification and alkalinization with up to 0.1 pH s(-1) (±0.002 pH s(-1)) rate of change across the dynamic range of our pH sensor (pH 4.5 to 7.5) in buffered solutions. Using dynamic current control, we generate and sustain 3 distinct pH microenvironments simultaneously to within ±0.04 pH for 13 minutes in a single solution, and we leverage these microenvironments to demonstrate spatially-resolved, pH-driven control of enzymatic activity. In addition to straightforward applications of spatio-temporal pH control (e.g. efficiently studying pH-dependencies of chemical interactions), the technique opens completely new avenues for implementing complex systems through dynamic control of enzyme activation, protein binding affinity, chemical reactivity, chemical release, molecular self-assembly, and many more pH-controlled processes.

Electrochemical Impedance Spectroscopy (EIS) is a powerful electrochemical technique to detect biomolecules. EIS has the potential of carrying out label-free and real-time detection, and in addition, can be easily implemented using electronic integrated circuits (ICs) that are built through standard semiconductor fabrication processes. This paper focuses on the various design and optimization aspects of EIS ICs, particularly the bio-to-semiconductor interface design. We discuss, in detail, considerations such as the choice of the electrode surface in view of IC manufacturing, surface linkers, and development of optimal bio-molecular detection protocols. We also report experimental results, using both macro- and micro-electrodes to demonstrate the design trade-offs and ultimately validate our optimization procedures.