Time-traceable micro-taggants for anti-counterfeiting and secure distribution of food and medicines.

This study presents an innovative solution for the enhanced tracking and security of pharmaceuticals through the development of microstructures incorporating environmentally responsive, coded microparticles. Utilizing maskless photolithography, we engineered these microparticles with a degradable masking layer with 30 µm thickness that undergoes controlled dissolution. Quantitative analysis revealed that the protective layer's degradation, monitored by red fluorescence intensity, diminishes predictably over 144 h in phosphate-buffered saline under physiological conditions. This degradation not only confirms the microparticles' integrity but also allows the extraction of encoded information, which can serve as a robust indicator of medicinal shelf life and a deterrent to tampering. These findings indicate the potential for applying this technology in real-time monitoring of pharmaceuticals, ensuring quality and authenticity in the supply chain.

Highly Accurate Sequence- and Position-Independent Error Profiling of DNA Synthesis and Sequencing.

A comprehensive error analysis of DNA-stored data during processing, such as DNA synthesis and sequencing, is crucial for reliable DNA data storage. Both synthesis and sequencing errors depend on the sequence and the transition of bases of nucleotides; ignoring either one of the error sources leads to technical challenges in minimizing the error rate. Here, we present a methodology and toolkit that utilizes an oligonucleotide library generated from a 10-base-shifted sequence array, which is individually labeled with unique molecular identifiers, to delineate and profile DNA synthesis and sequencing errors simultaneously. This methodology enables position- and sequence-independent error profiling of both DNA synthesis and sequencing. Using this toolkit, we report base transitional errors in both synthesis and sequencing in general DNA data storage as well as degenerate-base-augmented DNA data storage. The methodology and data presented will contribute to the development of DNA sequence designs with minimal error.

Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics.

Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.

Pen-drawn Marangoni swimmer.

Pen-drawing is an intuitive, convenient, and creative fabrication method for delivering emergent and adaptive design to real devices. To demonstrate the application of pen-drawing to robot construction, we developed pen-drawn Marangoni swimmers that perform complex programmed tasks using a simple and accessible manufacturing process. By simply drawing on substrates using ink-based Marangoni fuel, the swimmers demonstrate advanced robotic motions such as polygon and star-shaped trajectories, and navigate through maze. The versatility of pen-drawing allows the integration of the swimmers with time-varying substrates, enabling multi-step motion tasks such as cargo delivery and return to the original place. We believe that our pen-based approach will significantly expand the potential applications of miniaturized swimming robots and provide new opportunities for simple robotic implementations.

I-LIFT (image-based laser-induced forward transfer) platform for manipulating encoded microparticles.

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.

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches.

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

Photopatterned microswimmers with programmable motion without external stimuli.

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.

Advances in Tumor Sampling and Sequencing in Breast Cancer and their Application in Precision Diagnostics and Therapeutics.

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.

Direct 2D-to-3D transformation of pen drawings.

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.

Gradient-Wrinkled Microparticle with Grayscale Lithography Controlling the Cross-Linking Densities for High Security Level Anti-Counterfeiting Strategies.

Physical unclonable functions (PUFs) enable different characteristics according to the purpose, such as easy to access identification, high security level, and high code capacity, against counterfeiting a product. However, most multiplex approaches have been implemented by embedding several security features rather than one feature. In this paper, we present a high security level anti-counterfeiting strategy using only labyrinth wrinkle patterns with different complexities, which can be used as unique and unclonable codes. To generate codes with different levels in a microtaggant, we fabricated wrinkle patterns with characteristic wavelength gradients using grayscale lithography. The elastic modulus of the polymer substrate and corresponding wavelength after the wrinkling process were controlled by designing the gray level of each subcode region in a gray-level mask image for photopolymerization of the microparticle substrate. We then verified the uniqueness of the extracted minutia codes through a cross-correlation analysis. Finally, we demonstrated the authentication strategies by decoding different minutia codes according to the scanning resolution during the decoding. Overall, the presented patterning method can be widely used in security code generation.

A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System.

Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones-corresponding to ~14% of the library complexity-were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.

Microspinning: Local Surface Mixing via Rotation of Magnetic Microparticles for Efficient Small-Volume Bioassays.

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.

OPENchip: an on-chip in situ molecular profiling platform for gene expression analysis and oncogenic mutation detection in single circulating tumour cells.

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.

Barcode-free next-generation sequencing error validation for ultra-rare variant detection.

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 (< 1%) because of high sequencing errors (> 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.

One-Step Generation of a Drug-Releasing Hydrogel Microarray-On-A-Chip for Large-Scale Sequential Drug Combination Screening.

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.

Hierarchical shape-by-shape assembly of microparticles for micrometer-scale viral delivery of two different genes.

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.

ELIPatch, a thumbnail-size patch with immunospot array for multiplexed protein detection from human skin surface.

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-1ß are non-detectable in some cases. As demonstrations, we quantified cytokines from different skin regions and volunteers in a high-spatial-resolution.