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Encoded microparticles have great potential in small-volume multiplexed assays. It is important to link the micro-level assays to the macro-level by indexing and manipulating the microparticles to enhance their versatility. There are technologies to actively manipulate the encoded microparticles, but none is capable of directly manipulating the encoded microparticles with homogeneous physical properties. Here, we report the image-based laser-induced forward transfer system for active manipulation of the graphically encoded microparticles. By demonstrating the direct retrieval of the microparticles of interest, we show that this system has the potential to expand the usage of encoded microparticles.

We introduce highly programmable microscale swimmers driven by the Marangoni effect (Marangoni microswimmers) that can self-propel on the surface of water. Previous studies on Marangoni swimmers have shown the advantage of self-propulsion without external energy source or mechanical systems, by taking advantage of direct conversion from power source materials to mechanical energy. However, current developments on Marangoni microswimmers have limitations in their fabrication, thereby hindering their programmability and precise mass production. By introducing a photopatterning method, we generated Marangoni microswimmers with multiple functional parts with distinct material properties in high throughput. Furthermore, various motions such as time-dependent direction change and disassembly of swimmers without external stimuli are programmed into the Marangoni microswimmers., Maragoni microswimmers show the advantage of self-propulsion but their development is limited by fabrication techniques. Here, the authors use a photopatterning method which allows for a high throughput production of maragoni microswimmers with multiple functional parts and distinct materials properties.

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an

Alzheimer's disease (AD) is the most prevalent type of dementia. Reports have revealed that the peripheral immune system is linked to neuropathology; however, little is known about the contribution of B lymphocytes in AD. For this longitudinal study, 133 participants are included at baseline and second-year follow-up. Also, we analyze B cell receptor (BCR) repertoire data generated from a public dataset of three normal and 10 AD samples and perform BCR repertoire profiling and pairwise sharing analysis. As a result, longitudinal increase in B lymphocytes is associated with increased cerebral amyloid deposition and hyperactivates induced pluripotent stem cell-derived microglia with loss-of-function for beta-amyloid clearance. Patients with AD share similar class-switched BCR sequences with identical isotypes, despite the high somatic hypermutation rate. Thus, BCR repertoire profiling can lead to the development of individualized immune-based therapeutics and treatment. We provide evidence of both quantitative and qualitative changes in B lymphocytes during AD pathogenesis.

Analyzing archived peripheral blood smears is a potential route towards gaining cell morphology and genome information of blood cell types from various diseases. Yet, acquiring whole genome information from morphologically targeted cells was difficult, especially for rare cell types. The main causes for such difficulty were the inevitable usage of cell stains leading to whole genome amplification inhibition, and insufficient cell isolation performance of previously introduced laser microdissection (LMD) techniques. Here, we introduce a new laser-based cell isolation technique and a whole genome amplification (WGA) protocol optimized for whole genome analysis from minute input of hematologically stained cells. We were able to perform whole genome copy number profiling and SNP analysis from as little as 5 cells.

Intra- and Inter-tumoral heterogeneity is one of the main hurdles in diagnosing and treating breast cancer. Selecting, sampling, and sequencing the samples appropriately provide unique opportunities in realizing precision medicine. This chapter reviews some of the past landmarks, state-of-the-art technologies, and future directions of translational research in terms of tumor sampling technologies and sequencing in breast cancer. In the state-of-the-art technologies section, the technologies are categorized in terms of scientific, precision diagnostic, and precision therapeutic tools. Finally, limitations and future directions regarding various translational research for clinical applications using these technologies will be discussed.

Alzheimer’s disease (AD) is the most common type of dementia. Numerous reports have revealed that peripheral immune systems are linked to neuropathology; however, little is known about the contribution of B lymphocytes in AD. For this longitudinal study, one hundred-thirty-three participants were included in both baseline and follow-up 2nd year. Next, we performed next-generation sequencing (NGS)-based B cell receptor (BCR) repertoire profiling followed by pair-wise overlap analysis. The longitudinal change in the B lymphocytes population was associated with increased cerebral amyloid deposition. Furthermore, patients with AD shared highly similar class-switched BCR sequences with identical isotypes despite the high somatic hypermutation rate of BCR sequences. These commonalities of BCR repertoires of the patients with AD show the possibility of immunological stimuli by amyloid precursor protein (APP). Thus, we provide evidence for both quantitative and qualitative changes in B lymphocytes during AD pathogenesis. The genetic information of patients from the NGS-based BCR repertoire profiling can lead to the development of immune-based therapeutics and treatments for AD.

Technology for transforming a 2D pen drawing into a designed 3D structure enables highly accessible, rapid 3D fabrication., Pen drawing is a method that allows simple, inexpensive, and intuitive two-dimensional (2D) fabrication. To integrate such advantages of pen drawing in fabricating 3D objects, we developed a 3D fabrication technology that can directly transform pen-drawn 2D precursors into 3D geometries. 2D-to-3D transformation of pen drawings is facilitated by surface tension–driven capillary peeling and floating of dried ink film when the drawing is dipped into an aqueous monomer solution. Selective control of the floating and anchoring parts of a 2D precursor allowed the 2D drawing to transform into the designed 3D structure. The transformed 3D geometry can then be fixed by structural reinforcement using surface-initiated polymerization. By transforming simple pen-drawn 2D structures into complex 3D structures, our approach enables freestyle rapid prototyping via pen drawing, as well as mass production of 3D objects via roll-to-roll processing.

Complex oligonucleotide (oligo) libraries are essential materials for diverse applications in synthetic biology, pharmaceutical production, nanotechnology and DNA-based data storage. However, the error rates in synthesizing complex oligo libraries can be substantial, leading to increment in cost and labor for the applications. As most synthesis errors arise from faulty insertions and deletions, we developed a length-based method with single-base resolution for purification of complex libraries containing oligos of identical or different lengths. Our method-purification of multiplex oligonucleotide libraries by synthesis and selection-can be performed either step-by-step manually or using a next-generation sequencer. When applied to a digital data-encoded library containing oligos of identical length, the method increased the purity of full-length oligos from 83% to 97%. We also show that libraries encoding the complementarity-determining region H3 with three different lengths (with an empirically achieved diversity >106) can be simultaneously purified in one pot, increasing the in-frame oligo fraction from 49.6% to 83.5%.

Intra- and Inter-tumoral heterogeneity is one of the main hurdles in diagnosing and treating breast cancer. Selecting, sampling, and sequencing the samples appropriately provide unique opportunities in realizing precision medicine. This chapter reviews some of the past landmarks, state-of-the-art technologies, and future directions of translational research in terms of tumor sampling technologies and sequencing in breast cancer. In the state-of-the-art technologies section, the technologies are categorized in terms of scientific, precision diagnostic, and precision therapeutic tools. Finally, limitations and future directions regarding various translational research for clinical applications using these technologies will be discussed.

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have several technical limitations such as low process throughput from laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. TrueRepertoire™ provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment (scFv) library targeting human hepatocyte growth factor (hHGF) protein.

A revised WHO classification of hematopoietic neoplasm introduced the new category 'Myeloid Neoplasms with Germline Predisposition', reflecting the growing importance of genetic testing for myeloid neoplasms. Here, we investigated monozygotic twins with the same de novo mutation in GATA2 but different phenotypes. The patient suffering a bleeding tendency was diagnosed with myelodysplastic syndrome (MDS), and her monozygotic twin showed dysmegakaryopoietic features in the bone marrow. Targeted sequencing revealed the same germline mutation in GATA2, c.1192C T, in both sisters and different somatic mutations in 14 genes between the sisters. The GATA2 mutation was absent in both parents, and their hemograms were normal. The methylation profile of the GATA2 promoter region was different between the twins, showing denser promoter methylation in the patient, correlated with MDS. Thus, we concluded that the twins had acquired a de novo GATA2 mutation but showed different phenotypes, possibly due to the critical role of epigenetic changes.

DNA nanostructure-based mechanical systems that control the distance between elements of interest have demonstrated great potential for various applications, including nanoplasmonic systems, molecular reactors, and other nanotechnology platforms. However, previously reported systems could not collectively manipulate a 2D or 3D nanoscale network of elements to various forms in multiple stages. A reconfigurable DNA accordion rack structure is introduced that is a DNA beam lattice that changes its conformation with a small amount of short-length DNA locks as the controlling input. The lattice shape of the 2D DNA accordion rack and the diameter and the height of the 3D DNA nanotubular structure made of the DNA accordion rack could be controlled. Furthermore, by sequentially repeating the detachment and the attachment of the different DNA locks using strand displacement, the shape reconfiguration was repeatedly carried out.

DNA-based data storage has attracted attention because of its higher physical density of the data and longer retention time than those of conventional digital data storage1–7. However, previous DNA-based data storage lacked index features and the data quality of storage after a single access is not preserved, obstructing its industrial use. Here, we propose DNA micro-disks, quick response (QR)-coded micro-sized disks that harbour data-encoded DNA molecules for the efficient management of DNA-based data storage. We demonstrate the two major features that previous DNA-based data storage studies could not achieve. One feature is accessing data items efficiently by indexing the data-encoded DNA library. Another is achieving write-once-read-many (WORM) memory through the immobilization of DNA molecules on the disk and their enrichment through in situ DNA production. Through these features, the reliability of DNA-based data storage was increased by allowing multiple accession of data-encoded DNA without data loss.

Liquid biopsy holds promise towards practical implementation of personalized theranostics of cancer. In particular, circulating tumour cells (CTCs) can provide clinically actionable information that can be directly linked to prognosis or therapy decisions. In this study, gene expression patterns and genetic mutations in single CTCs are simultaneously analysed by strategically combining microfluidic technology and in situ molecular profiling technique. Towards this, the development and demonstration of the OPENchip (On-chip Post-processing ENabling chip) platform for single CTC analysis by epithelial CTC enrichment and subsequent in situ molecular profiling is reported. For in situ molecular profiling, padlock probes that identify specific desired targets to examine biomarkers of clinical relevance in cancer diagnostics were designed and used to create libraries of rolling circle amplification products. We characterize the OPENchip in terms of its capture efficiency and capture purity, and validate the probe design using different cell lines. By integrating the obtained results, molecular analyses of CTCs from metastatic breast cancer (HER2 (ERBB2) gene expression and PIK3CA mutations) and metastatic pancreatic cancer (KRAS gene mutations) patients were demonstrated without any off-chip processes. The results substantiate the potential implementation of early molecular detection of cancer through sequencing-free liquid biopsy.

The need for high-throughput screening has led to the miniaturization of the reaction volume of the chamber in bioassays. As the reactor gets smaller, surface tension dominates the gravitational or inertial force, and mixing efficiency decreases in small-scale reactions. Because passive mixing by simple diffusion in tens of microliter-scale volumes takes a long time, active mixing is needed. Here, we report an efficient micromixing method using magnetically rotating microparticles with patterned magnetization induced by magnetic nanoparticle chains. Because the microparticles have magnetization patterning due to fabrication with magnetic nanoparticle chains, the microparticles can rotate along the external rotating magnetic field, causing micromixing. We validated the reaction efficiency by comparing this micromixing method with other mixing methods such as simple diffusion and the use of a rocking shaker at various working volumes. This method has the potential to be widely utilized in suspension assay technology as an efficient mixing strategy.

The use of light in biotechnology has reversely enlightened the path for development in biosciences. In this chapter, the basic optical principles used in micro/nano systems for biological applications are explained and their applications are followed. This chapter is comprised of two parts: integration of optics in biodevices and optofluidics. The former part describes optics, fluorescence, and plasmonics that are integrated in micro/nano systems and related technology and discuss their current and future applications. The latter part describes fabrication and manipulation methods that are enabled by optics.

DNA-based data storage has emerged as a promising method to satisfy the exponentially increasing demand for information storage. However, practical implementation of DNA-based data storage remains a challenge because of the high cost of data writing through DNA synthesis. Here, we propose the use of degenerate bases as encoding characters in addition to A, C, G, and T, which augments the amount of data that can be stored per length of DNA sequence designed (information capacity) and lowering the amount of DNA synthesis per storing unit data. Using the proposed method, we experimentally achieved an information capacity of 3.37 bits/character. The demonstrated information capacity is more than twice when compared to the highest information capacity previously achieved. The proposed method can be integrated with synthetic technologies in the future to reduce the cost of DNA-based data storage by 50%.

The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants ( 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10−6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies., Next generation sequencing has difficulty in detecting rare-frequency variants due to high sequencing errors. Here the authors present a barcode-free error validation method that physically extracts erroneous reads to identify true variants.

Large‐scale screening of sequential drug combinations, wherein the dynamic rewiring of intracellular pathways leads to promising therapeutic effects and improvements in quality of life, is essential for personalized medicine to ensure realistic cost and time requirements and less sample consumption. However, the large‐scale screening requires expensive and complicated liquid handling systems for automation and therefore lowers the accessibility to clinicians or biologists, limiting the full potential of sequential drug combinations in clinical applications and academic investigations. Here, a miniaturized platform for high‐throughput combinatorial drug screening that is “pipetting‐free” and scalable for the screening of sequential drug combinations is presented. The platform uses parallel and bottom‐up formation of a heterogeneous drug‐releasing hydrogel microarray by self‐assembly of drug‐laden hydrogel microparticles. This approach eliminates the need for liquid handling systems and time‐consuming operation in high‐throughput large‐scale screening. In addition, the serial replacement of the drug‐releasing microarray‐on‐a‐chip facilitates different drug exchange in each and every microwell in a simple and highly parallel manner, supporting scalable implementation of multistep combinatorial screening. The proposed strategy can be applied to various forms of combinatorial drug screening with limited amounts of samples and resources, which will broaden the use of the large‐scale screening for precision medicine.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to ångström resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration. In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

Introductory paragraphDNA-based data storage has emerged as a promising method to satisfy the exponentially increasing demand for information storage. However, practical implementation of DNA-based data storage remains a challenge because of the high cost of DNA per unit data. Here, we propose the use of eleven degenerate bases as encoding characters in addition to A, C, G, and T, which increases the information capacity (the amount of data that can be stored per length of DNA sequence designed) and reduce the cost of DNA per unit data. Using the proposed method, we experimentally achieved an information capacity of 3.37 bits/character, which is more than twice when compared to the highest information capacity previously achieved. Finally, the platform was projected to reduce the cost of DNA-based data storage by 50%.

Proteins secreted by skin have great potential as biomarkers for interpreting skin conditions. However, inconvenience in handling and bulky size of existing methods are existing limitations. Here, we describe a thumb-nail sized patch with the array of microdisks which captures multiple proteins from the skin surface. Microdisks with antibody on the surface enable multiplexed immunoassay. By self-assembly, microdisks are placed into 2-dimensional arrays on adhesive tape. The proposed Enzyme-Linked Immunospot array on a Patch shows sufficient sensitivity for IL-1α, IL1RA, IL-17A, IFN-g, and TNF-α, while IL-6 and IL-1β are non-detectable in some cases. As demonstrations, we quantified cytokines from different skin regions and volunteers in a high-spatial-resolution.

A spatially resolved analysis of the heterogeneous cancer genome, in which the data are connected to the three-dimensional space of a tumour, is crucial to understand cancer biology and the clinical impact of cancer heterogeneity on patients. However, despite recent progress in spatially resolved transcriptomics, spatial mapping of genomic data in a high-throughput and high-resolution manner has been challenging due to current technical limitations. Here, we describe a novel approach, phenotype-based high-throughput laser-aided isolation and sequencing (PHLI-seq), which enables high-throughput isolation of a single-cell or a small number of cells and their genome-wide sequence analysis to construct genomic maps within cancer tissue in relation to the phenotypes of the cells. By applying PHLI-seq, we reveal the heterogeneity of breast cancer tissues at a high resolution and map the genomic landscape of the cells to their corresponding spatial locations and phenotypes in the tumour mass. Additionally, with different staining modalities, the genotypes of the cells can be connected to corresponding phenotypic information of the tissue. Together with the spatially resolved genomic analysis, we can infer the histories of heterogeneous cancer cells in two or three dimensions, providing significant insight into cancer biology and precision medicine.

Circulating tumor cells (CTCs) are well known for causing metastasis in the cancer patients because the escaped CTCs from the primary tumor site move to the distant organs through the blood vessels. Thus, the genetic information inside the CTCs is exclusively studied to extract out clinical values of them. Because of such values of CTCs, there have been efforts to detect the CTCs from the patient blood. But only few platforms are available to analyze the whole genome of the CTCs with high resolution after the separation of the CTCs. In this study, we applied Laser-induced Isolation of Microstructure On transferrable-chip and sequencing (LIMO-seq) to retrieve the CTCs and sequence the whole genome using next generation sequencing (NGS). With the simple photo-lithography process, we designed the biochip consisting of the separable microstructures and applied LIMO-seq to solve the current limitations of the single CTC genome analysis. Using the chip, we showed that the CTCs captured by the immune-affinity reaction were readily immuno-fluorescently stained and isolated at a single cell level from the chip using LIMO-seq. We analyzed the whole genome sequences of the isolated single cells for the molecular profiling using NGS technology. Furthermore, we isolated molecule-labelled single CTCs from a breast cancer patient's whole blood to demonstrate clinical potential of this platform. The isolation of the captured CTCs with LIMO-seq will enable CTC-based diagnostics for treating and monitoring the cancer patients.

Single cell analysis of heterogeneous circulating tumor cells (CTCs), by which the genomic profiles of rare single CTCs are connected to the clinical status of cancer patients, is crucial for understanding cancer metastasis and the clinical impact on patients. However, the heterogeneity in genotypes and phenotypes and rarity of CTCs have limited extensive single CTC genome research, further hindering clinical investigation. Despite recent efforts to build platforms that separate CTCs, the investigation on CTCs is difficult due to the lack of a retrieval process at the single cell level. In this study, laser-induced isolation of microstructures on an optomechanically-transferrable-chip and sequencing (LIMO-seq) is applied for whole genome sequencing of single CTCs. Also, the whole genome sequences and the molecular profiles of the isolated single cells from the whole blood of a breast cancer patient are analyzed.

Recent advances in biochip technologies that connect next-generation sequencing (NGS) to real-world problems have facilitated breakthroughs in science and medicine. Because biochip technologies are themselves used in sequencing technologies, the main strengths of biochips lie in their scalability and throughput. Through the advantages of biochips, NGS has facilitated groundbreaking scientific discoveries and technical breakthroughs in medicine. However, all current NGS platforms require nucleic acids to be prepared in a certain range of concentrations, making it difficult to analyze biological systems of interest. In particular, many of the most interesting questions in biology and medicine, including single-cell and rare-molecule analysis, require strategic preparation of biological samples in order to be answered. Answering these questions is important because each cell is different and exists in a complex biological system. Therefore, biochip platforms for single-cell or rare-molecule analyses by NGS, which allow convenient preparation of nucleic acids from biological systems, have been developed. Utilizing the advantages of miniaturizing reaction volumes of biological samples, biochip technologies have been applied to diverse fields, from single-cell analysis to liquid biopsy. From this perspective, here, we first review current state-of-the-art biochip technologies, divided into two broad categories: microfluidic- and micromanipulation-based methods. Then, we provide insights into how future biochip systems will aid some of the most important biological and medical applications that require NGS. Based on current and future biochip technologies, we envision that NGS will come ever closer to solving more real-world scientific and medical problems.

Understanding tissue engineering using a bottom-up approach has been hindered by technical limitations because no platform can demonstrate the controlled formation of a heterogeneous population of cells in microscale. Here, we demonstrate hierarchical shape-by-shape assembly of virus-laden particles into larger ones to transfect two different genes on the seeded cells. We show that smaller daughter particles with different sizes and shapes can be assembled into the matching indentations of larger parent particles with different sizes and shapes. Then, we transfected a population of cells with two different gene-transfecting viruses, each of which was laden on the parent or daughter particles.

Math discussions are important when learning math. Explaining one’s thinking, listening to other’s thoughts, and reflecting are but a few of the benefits derived from discussions held in class. However, with the growth of online courses, how do math discussions change when in an online setting? While much research exists about math discussions in classrooms, there is not much research on math discussions held online. Due to the important role of discussions in learning math, along with the growing trend of online classes, this study begins to take a look at how students make sense of and keep track of each other’s comments in an online discussion. In these online discussions, turn taking is not as intuitive as face-to-face interactions. Making sense of the discussion sequence and theme can also be challenging. In this study, I found that students used terms that represented mathematical operations to better explain their thought processes and also kept track of how their peers used these terms as well. These findings suggest that, for these students, when in an online discussion, the terms used were of importance when trying to make their thinking clear to their classmates. Also, in these groups, the mathematical terms were commonly used and re-used by more than one individual in trying to gain a consensus in their group thinking. These findings are important when thinking about how to best foster math discussion and learning in an online environment and for designing online classes that institutions use to supplement or support students.

Integrating the microfluidic device with the optomechanical system, we developed a novel rare cell isolation biochip with serparable microstructures. Rare cells such as circulating tumor cells (CTCs) are well-known for causing metastasis in the cancer patients. These CTCs were captured using immune-affinity method with the epithelial cell adhesion molecule binding antibody (Anti-EpCAM Antibody) on SU-8 microstructure fabricated on the slide glass via photolithography. Here, we demostrated that CTCs captured on SU-8 microstructure, and isolated the structures from the biochip to micro tubes using nanosecond infrared laser pulse for further analysis such as PCR, RT-qPCR and NGS.