Plasmonic Optical Wells-Based Enhanced Rate PCR.

Rapid, sensitive, inexpensive point-of-care molecular diagnostics are crucial for the efficient control of spreading viral diseases and biosecurity of global health. However, the gold standard, polymerase chain reaction (PCR) is time-consuming and expensive and needs specialized testing laboratories. Here, we report a low-cost yet fast, selective, and sensitive Plasmonic Optical Wells-Based Enhanced Rate PCR: POWER-PCR. We optimized the efficient optofluidic design of 3D plasmonic optical wells via the computational simulation of light-to-heat conversion and thermophoretic convection in a self-created plasmonic cavity. The POWER-PCR chamber with a self-passivation layer can concentrate incident light to accumulate molecules, generate rapid heat transfer and thermophoretic flow, and minimize the quenching effect on the naked Au surface. Notably, we achieved swift photothermal cycling of nucleic acid amplification in POWER-PCR on-a-chip in 4 min 24 s. The POWER-PCR will provide an excellent solution for affordable and sensitive molecular diagnostics for precision medicine and preventive global healthcare.

Spontaneous Particle Ordering, Sorting, and Assembly on Soap Films.

Soap bubbles exhibit abundant fascinating phenomena throughout the entire life of evolution with different fundamental physics governing them. Nevertheless, the complicated dynamics of small objects in soap films are still unrevealed. Here, we report the first observation of spontaneous particle ordering in a complicated galaxy of soap films without any external energy. The balance of interfacial tension at two liquid-gas interfaces is theoretically predicted to govern belted wetted particles (BWPs) traveling along a specified path spontaneously. Such spontaneous particle path-finding is found to depend on the particle size and hydrophilic properties. Spontaneous particle sorting is directly realized via these discrete and distinctive paths for different particles. The deformation of the soap membrane facilitates 1D/2D particle organization along the path. This observation represents the discovery of a new spontaneous order phenomenon in soap film systems and provides a new energy-free approach for particle separation and soft colloidal crystal assembly.

Exosome detection via the ultrafast-isolation system: EXODUS.

Exosomes have shown great potential in disease diagnostics and therapeutics. However, current isolation approaches are burdensome and suffer from low speed, yield and purity, limiting basic research and clinical applications. Here, we describe an efficient exosome detection method via the ultrafast-isolation system (EXODUS) that allows automated label-free purification of exosomes from varied biofluids. We obtained the ultra-efficient purification of exosomes by negative pressure oscillation and double coupled harmonic oscillator-enabled membrane vibration. Our two coupled oscillators generate dual-frequency transverse waves on the membranes, enabling EXODUS to outperform other isolation techniques in speed, purity and yield. We demonstrated EXODUS by purifying exosomes from urine samples of 113 patients and validated the practical relevance in exosomal RNA profiling with the high-resolution capability and high-throughput analysis.

The genetic source tracking of human urinary exosomes.

The genetic origins of nanoscale extracellular vesicles in our body fluids remains unclear. Here, we perform a tracking analysis of urinary exosomes via RNA sequencing, revealing that urine exosomes mostly express tissue-specific genes for the bladder and have close cell-genetic relationships to the endothelial cell, basal cell, monocyte, and dendritic cell. Tracking the differentially expressed genes of cancers and corresponding enrichment analysis show urine exosomes are intensively involved in immune activities, indicating that they may be harnessed as reliable biomarkers of noninvasive liquid biopsy in cancer genomic diagnostics and precision medicine.

Harmonic acoustics for dynamic and selective particle manipulation.

Precise and selective manipulation of colloids and biological cells has long been motivated by applications in materials science, physics and the life sciences. Here we introduce our harmonic acoustics for a non-contact, dynamic, selective (HANDS) particle manipulation platform, which enables the reversible assembly of colloidal crystals or cells via the modulation of acoustic trapping positions with subwavelength resolution. We compose Fourier-synthesized harmonic waves to create soft acoustic lattices and colloidal crystals without using surface treatment or modifying their material properties. We have achieved active control of the lattice constant to dynamically modulate the interparticle distance in a high-throughput (>100 pairs), precise, selective and reversible manner. Furthermore, we apply this HANDS platform to quantify the intercellular adhesion forces among various cancer cell lines. Our biocompatible HANDS platform provides a highly versatile particle manipulation method that can handle soft matter and measure the interaction forces between living cells with high sensitivity.

Engineering in Medicine To Address the Challenge of Cancer Drug Resistance: From Micro- and Nanotechnologies to Computational and Mathematical Modeling.

Drug resistance has profoundly limited the success of cancer treatment, driving relapse, metastasis, and mortality. Nearly all anticancer drugs and even novel immunotherapies, which recalibrate the immune system for tumor recognition and destruction, have succumbed to resistance development. Engineers have emerged across mechanical, physical, chemical, mathematical, and biological disciplines to address the challenge of drug resistance using a combination of interdisciplinary tools and skill sets. This review explores the developing, complex, and under-recognized role of engineering in medicine to address the multitude of challenges in cancer drug resistance. Looking through the "lens" of intrinsic, extrinsic, and drug-induced resistance (also referred to as "tolerance"), we will discuss three specific areas where active innovation is driving novel treatment paradigms: (1) nanotechnology, which has revolutionized drug delivery in desmoplastic tissues, harnessing physiochemical characteristics to destroy tumors through photothermal therapy and rationally designed nanostructures to circumvent cancer immunotherapy failures, (2) bioengineered tumor models, which have benefitted from microfluidics and mechanical engineering, creating a paradigm shift in physiologically relevant environments to predict clinical refractoriness and enabling platforms for screening drug combinations to thwart resistance at the individual patient level, and (3) computational and mathematical modeling, which blends in silico simulations with molecular and evolutionary principles to map mutational patterns and model interactions between cells that promote resistance. On the basis that engineering in medicine has resulted in discoveries in resistance biology and successfully translated to clinical strategies that improve outcomes, we suggest the proliferation of multidisciplinary science that embraces engineering.

Quantum Electrodynamic Behavior of Chlorophyll in a Plasmonic Nanocavity.

Plasmonic nanocavities have been used as a novel platform for studying strong light-matter coupling, opening access to quantum chemistry, material science, and enhanced sensing. However, the biomolecular study of cavity quantum electrodynamics (QED) is lacking. Here, we report the quantum electrodynamic behavior of chlorophyll-a in a plasmonic nanocavity. We construct an extreme plasmonic nanocavity using Au nanocages with various linker molecules and Au mirrors to obtain a strong coupling regime. Plasmon resonance energy transfer (PRET)-based hyperspectral imaging is applied to study the electrodynamic behaviors of chlorophyll-a in the nanocavity. Furthermore, we observe the energy level splitting of chlorophyll-a, similar to the cavity QED effects due to the light-matter interactions in the cavity. Our study will provide insight for further studies in quantum biological electron or energy transfer, electrodynamics, the electron transport chain of mitochondria, and energy harvesting, sensing, and conversion in both biological and biophysical systems.

Inner-Membrane-Bound Gold Nanoparticles as Efficient Electron Transfer Mediators for Enhanced Mitochondrial Electron Transport Chain Activity.

Electron transfer through the mitochondrial electron transport chain (ETC) can be critically blocked by the dysfunction of protein complexes. Redox-active molecules have been used to mediate the electron transfer in place of the dysfunctional complexes; however, they are limited to replacing complex I and are known to be toxic. Here we report artificial mitochondrial electron transfer pathways that enhance ETC activity by exploiting inner-membrane-bound gold nanoparticles (GNPs) as efficient electron transfer mediators. The hybridization of mitochondria with GNPs, driven by electrostatic interaction, is successfully visualized in real time at the level of a single mitochondrion. By observing quantized quenching dips via plasmon resonance energy transfer, we reveal that the hybridized GNPs are bound to the inner membrane of mitochondria irrespective of the presence of the outer membrane. The ETC activity of mitochondria with GNPs such as membrane potential, oxygen consumption, and ATP production is remarkably increased in vitro.

Integrated Point-of-Care Molecular Diagnostic Devices for Infectious Diseases.

The global outbreaks of deadly infectious diseases caused by pathogenic microorganisms have threatened public health worldwide and significantly motivated scientists to satisfy an urgent need for a rapid and accurate detection of pathogens. Traditionally, the culture-based technique is considered as the gold standard for pathogen detection, yet it has a long turnaround time due to the overnight culturing and pathogen isolation. Alternatively, nucleic acid amplification tests provide a relatively shorter turnaround time to identify whether pathogens exist in individuals with high sensitivity and high specificity. In most cases, nucleic acid amplification tests undergo three steps: sample preparation, nucleic acid amplification, and signal transduction. Despite the explosive advancement in nucleic acid amplification and signal transduction technologies, the complex and labor-intensive sample preparation steps remain a bottleneck to create a transformative integrated point-of-care (POC) molecular diagnostic device. Researchers have attempted to simplify and integrate the sample preparations for nucleic acid-based molecular diagnostic devices with innovative progress in integration strategies, engineered materials, reagent storages, and fluid actuation. Therefore, understanding the know-how and obtaining truthful knowledge of existing integrated POC molecular diagnostic devices comprising sample preparations, nucleic acid amplification, and signal transduction can generate innovative solutions to achieve personalized precision medicine and improve global health.In this Account, we discuss the challenges of automated sample preparation solutions integrated with nucleic acid amplification and signal transduction for rapid and precise home diagnostics. Blood, nasal swab, saliva, urine, and stool are emphasized as the most commonly used clinical samples for integrated POC molecular diagnostics of infectious diseases. Even though these five types of samples possess relatively correlated biomarkers due to the human body's circulatory system, each shows unique properties and exclusive advantages for molecular diagnostics in specific situations, which are included in this Account. We examine different integrated POC devices for sample preparation, which includes pathogen isolation and enrichment from the crude sample and nucleic acid purification from isolated pathogens. We present the promising on-chip integration approaches for nucleic acid amplification. We also investigate the on-chip integration methods for reagent storage, which is crucial to simplify the manual operation for end-users. Finally, we present several integrated POC molecular diagnostic devices for infectious diseases. The integrated sample preparation and nucleic acid amplification approach reviewed here can potentially impact the next generation of POC molecular home diagnostic chips, which will significantly impact public health, emergency medicine, and global biosecurity.

Electropolymerized-molecularly imprinted polymers (E-MIPS) as sensing elements for the detection of dengue infection.

A straightforward in situ detection method for dengue infection was demonstrated through the molecular imprinting of a dengue nonstructural protein 1 (NS1) epitope into an electropolymerized molecularly imprinted polyterthiophene (E-MIP) film sensor. The key enabling step in the sensor fabrication is based on an epitope imprinting strategy, in which short peptide sequences derived from the original target molecules were employed as the main template for detection and analysis. The formation of the E-MIP sensor films was facilitated using cyclic voltammetry (CV) and monitored in situ by electrochemical quartz crystal microbalance (EC-QCM). Surface properties were analyzed using different techniques including atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and polarization modulation-infrared reflection-adsorption (PM-IRRAS). The standard calibration curve (R = 0.9830) was generated for the detection of the epitope, Ac-VHTWTEQYKFQ-NH2, with a linear range of 0.2 to 30 µg/mL and detection limit of 0.073 µg/mL. A separate calibration curve (R = 0.9786) was obtained using spiked buffered solutions of dengue NS1 protein, which resulted in a linear range of 0.2 to 10 µg/mL and a detection limit of 0.056 µg/mL. The fabricated E-MIP sensor exhibited long-term stability, high sensitivity, and good selectivity towards the targeted molecules. These results indicated that the formation of the exact and stable cavity imprints in terms of size, shape, and functionalities was successful. In our future work, we aim to use our E-MIP sensors for NS1 detection in real-life samples such as serum and blood.

Vancomycin-conjugated polydopamine-coated magnetic nanoparticles for molecular diagnostics of Gram-positive bacteria in whole blood.

BACKGROUND: Sepsis is caused mainly by infection in the blood with a broad range of bacterial species. It can be diagnosed by molecular diagnostics once compounds in the blood that interfere with molecular diagnostics are removed. However, this removal relies on ultracentrifugation. Immunomagnetic separation (IMS), which typically uses antibody-conjugated silica-coated magnetic nanoparticles (Ab-SiO2-MNPs), has been widely applied to isolate specific pathogens in various types of samples, such as food and environmental samples. However, its direct use in blood samples containing bacteria is limited due to the aggregation of SiO2-MNPs in the blood and inability to isolate multiple species of bacteria causing sepsis. RESULTS: In this study, we report the synthesis of vancomycin-conjugated polydopamine-coated (van-PDA-MNPs) enabling preconcentration of multiple bacterial species from blood without aggregation. The presence of PDA and van on MNPs was verified using transmission electron microscopy, X-ray photoelectron spectroscopy, and energy disruptive spectroscopy. Unlike van-SiO2-MNPs, van-PDA-MNPs did not aggregate in the blood. Van-PDA-MNPs were able to preconcentrate several species of Gram-positive bacteria in the blood, lowering the limit of detection (LOD) to 10 colony forming units/mL by polymerase chain reaction (PCR) and quantitative PCR (qPCR). This is 10 times more sensitive than the LOD obtained by PCR and qPCR using van-SiO2-MNPs. CONCLUSION: These results suggest that PDA-MNPs can avoid aggregation in blood and be conjugated with receptors, thereby improving the sensitivity of molecular diagnostics of bacteria in blood samples.

Uncovering the Metabolic Origin of Aspartate for Tumor Growth Using an Integrated Molecular Deactivator.

Reprogrammed glucose metabolism is vital for cancer cells, but aspartate, an intermediate metabolic product, is the limiting factor for cancer cell proliferation. However, due to the complexity of metabolic pathways, it remains unclear whether glucose is the primary source of endogenous aspartate. Here, we report the design of an innovative molecular deactivator, based on a multifunctional upconversion nanoprobe, to explore the link between glucose and aspartate. This molecular deactivator mainly works in the acidic, hypoxic tumor microenvironment and deactivates multiple types of glucose transporters on cancer cell membranes upon illumination at 980 nm. Cancer cell proliferation in vivo is strongly inhibited by blocking glucose transporters. Our experimental data confirm that the cellular synthesis of aspartate for tumor growth is glucose-dependent. This work also demonstrates the untapped potential of molecularly engineered upconversion nanoprobes for discovering hidden metabolic pathways and improving therapeutic efficacy of conventional antitumor drugs.

Art-on-a-Chip: Preserving Microfluidic Chips for Visualization and Permanent Display.

"After a certain high level of technical skill is achieved, science and art tend to coalesce in aesthetics, plasticity, and form. The greatest scientists are always artists as well." said Albert Einstein. Currently, photographic images bridge the gap between microfluidic/lab-on-a-chip devices and art. However, the microfluidic chip itself should be a form of art. Here, novel vibrant epoxy dyes are presented in combination with a simple process to fill and preserve microfluidic chips, to produce microfluidic art or art-on-a-chip. In addition, this process can be used to produce epoxy dye patterned substrates that preserve the geometry of the microfluidic channels-height within 10% of the mold master. This simple approach for preserving microfluidic chips with vibrant, colorful, and long-lasting epoxy dyes creates microfluidic chips that can easily be visualized and photographed repeatedly, for at least 11 years, and hence enabling researchers to showcase their microfluidic chips to potential graduate students, investors, and collaborators.

Active Surface Hydrophobicity Switching and Dynamic Interfacial Trapping of Microbial Cells by Metal Nanoparticles for Preconcentration and In-Plane Optical Detection.

The surface hydrophobicity of a microbial cell is known to be one of the important factors in its adhesion to an interface. To date, such property has been altered by either genetic modification or external pH, temperature, and nutrient control. Here we report a new strategy to engineer a microbial cell surface and discover the unique dynamic trapping of hydrophilic cells at an air/water interface via hydrophobicity switching. We demonstrate the surface transformation and hydrophobicity switching of Escherichia coli (E. coli) by metal nanoparticles. By employing real-time dark-field imaging, we directly observe that hydrophobic gold nanoparticle-coated E. coli, unlike its naked counterpart, is irreversibly trapped at the air/water interface because of elevated hydrophobicity. We show that our surface transformation method and resulting dynamic interfacial trapping can be generally extended to Gram-positive bateria, Gram-negative bacteria, and fungi. As the dynamic interfacial trapping allows the preconcentration of microbial cells, high intensity of scattering light, in-plane focusing, and near-field enhancement, we are able to directly quantify E. coli as low as 1.0 × 103 cells/ml by using a smartphone with an image analyzer. We also establish the identification of different microbial cells by the characteristic Raman transitions directly measured from the interfacially trapped cells.

Transcriptomes of lineage-specific Drosophila neuroblasts profiled by genetic targeting and robotic sorting.

A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates.

Molecular profiling of single circulating tumor cells from lung cancer patients.

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

Asymmetric Nanopore Electrode-Based Amplification for Electron Transfer Imaging in Live Cells.

Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Single-Step LRET Aptasensor for Rapid Mycotoxin Detection.

Contamination of foods by mycotoxins is a common yet serious problem. Owing to the increase in consumption of fresh produce, consumers have become aware of food safety issues caused by mycotoxins. Therefore, rapid and sensitive mycotoxin detection is in great demand in fields such as food safety and public health. Here we report a single-step luminescence resonance energy transfer (LRET) aptasensor for mycotoxin detection. To accomplish the single-step sensor, our sensor was constructed by linking a quencher-labeled aptamer through a linker to the surface of upconversion nanoparticles (UCNPs). Our LRET aptasensor is composed of Mn2+-doped NaYF4:Yb3+,Er3+ UCNPs as the LRET donor, and black hole quencher 3 (BHQ3) as the acceptor. The maximum quenching efficiency is obtained by modulating the linker length, which controls the distance between the quencher and the UCNPs. Our distinctive design of LRET aptasensor allows detection of mycotoxins selectively in colored food samples within 10 min without multiple bioassay steps. We believe our single-step aptasensor has a significant potential for on-site detection of food contaminants, environmental pollutants, and biological metabolites.