Digital direct detection of microRNAs using single molecule arrays.

MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.

DNA materials: bridging nanotechnology and biotechnology.

CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a DNA-protein conjugate as a universal adapter for protein detection. We further demonstrate a diverse assortment of applications for these DNA materials including diagnostics, protein production, controlled drug release systems, the exploration of life evolution, and plasmonics. Although DNA has shown great potential as both substrate and linker in the construction of DNA materials, it is still in the initial stages of becoming a well-established and widely used material. Important challenges include the ease of design and fabrication, scaling-up, and minimizing cost. We envision that DNA materials will continue to bridge the gap between nanotechnology and biotechnology and will ultimately be employed for many real-world applications.

A universal DNA-based protein detection system.

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

Using Next-Generation Sequencing to Explore Genetics and Race in the High School Classroom.

With the development of new sequencing and bioinformatics technologies, concepts relating to personal genomics play an increasingly important role in our society. To promote interest and understanding of sequencing and bioinformatics in the high school classroom, we developed and implemented a laboratory-based teaching module called "The Genetics of Race." This module uses the topic of race to engage students with sequencing and genetics. In the experimental portion of this module, students isolate their own mitochondrial DNA using standard biotechnology techniques and collect next-generation sequencing data to determine which of their classmates are most and least genetically similar to themselves. We evaluated the efficacy of this module by administering a pretest/posttest evaluation to measure student knowledge related to sequencing and bioinformatics, and we also conducted a survey at the conclusion of the module to assess student attitudes. Upon completion of our Genetics of Race module, students demonstrated significant learning gains, with lower-performing students obtaining the highest gains, and developed more positive attitudes toward scientific research.

Personal microbiomes and next-generation sequencing for laboratory-based education.

Sequencing and bioinformatics technologies have advanced rapidly in recent years, driven largely by developments in next-generation sequencing (NGS) technology. Given the increasing importance of these advances, there is a growing need to incorporate concepts and practices relating to NGS into undergraduate and high school science curricula. We believe that direct access to sequencing and bioinformatics will improve the ability of students to understand the information obtained through these increasingly ubiquitous research tools. In this commentary, we discuss approaches and challenges for bringing NGS into the classroom based on our experiences in developing and running a microbiome project in high school and undergraduate courses. We describe strategies for maximizing student engagement through establishing personal relevance and utilizing an inquiry-based structure. Additionally, we address the practical issues of incorporating cutting edge technologies into an established curriculum. Looking forward, we anticipate that NGS educational experiments will become more commonplace as sequencing costs continue to decrease and the workflow becomes more user friendly.

Point-of-care nucleic acid detection using nanotechnology.

Recent developments in nanotechnology have led to significant advancements in point-of-care (POC) nucleic acid detection. The ability to sense DNA and RNA in a portable format leads to important applications for a range of settings, from on-site detection in the field to bedside diagnostics, in both developing and developed countries. We review recent innovations in three key process components for nucleic acid detection: sample preparation, target amplification, and read-out modalities. We discuss how the advancements realized by nanotechnology are making POC nucleic acid detection increasingly applicable for decentralized and accessible testing, in particular for the developing world.

Enhanced transcription and translation in clay hydrogel and implications for early life evolution.

In most contemporary life forms, the confinement of cell membranes provides localized concentration and protection for biomolecules, leading to efficient biochemical reactions. Similarly, confinement may have also played an important role for prebiotic compartmentalization in early life evolution when the cell membrane had not yet formed. It remains an open question how biochemical reactions developed without the confinement of cell membranes. Here we mimic the confinement function of cells by creating a hydrogel made from geological clay minerals, which provides an efficient confinement environment for biomolecules. We also show that nucleic acids were concentrated in the clay hydrogel and were protected against nuclease, and that transcription and translation reactions were consistently enhanced. Taken together, our results support the importance of localized concentration and protection of biomolecules in early life evolution, and also implicate a clay hydrogel environment for biochemical reactions during early life evolution.

High-yield cell-free protein production from P-gel.

Cell-free systems represent a promising approach to quickly and easily produce preparative amounts of proteins. However, it is still challenging to obtain high volumetric yields (>mg ml(-1)) of proteins from the present cell-free systems. This protocol presents a cell-free protein synthesis method using a novel DNA gel that dramatically increases protein yield compared with current systems. This protein-producing gel (termed 'P-gel system' or 'P-gel'), which consists of genes as part of the gel scaffolding, can produce mg ml(-1) amounts of functional proteins. This protocol describes steps pertaining to plasmid design, fabrication of P-gel molds, formation of P-gel micropads and cell-free protein expression with an expected yield of up to 5 mg ml(-1) of functional Renilla luciferase (Rluc). This entire process can take 1-3 d, depending on the desired quantity of protein.

Nanopatterning self-assembled nanoparticle superlattices by moulding microdroplets.

Highly ordered arrays of nanoparticles exhibit many properties that are not found in their disordered counterparts. However, these nanoparticle superlattices usually form in a far-from-equilibrium dewetting process, which precludes the use of conventional patterning methods owing to a lack of control over the local dewetting dynamics. Here, we report a simple yet efficient approach for patterning such superlattices that involves moulding microdroplets containing the nanoparticles and spatially regulating their dewetting process. This approach can provide rational control over the local nucleation and growth of the nanoparticle superlattices. Using DNA-capped gold nanoparticles as a model system, we have patterned nanoparticle superlattices over large areas into a number of versatile structures with high degrees of internal order, including single-particle-width corrals, single-particle-thickness microdiscs and submicrometre-sized 'supra-crystals'. Remarkably, these features could be addressed by micropatterned electrode arrays, suggesting potential applications in bottom-up nanodevices.