Naked-Eye LAMP Assay of M. tuberculosis in Sputum by In Situ Au Nanoprobe Identification: For the In Vitro Diagnostics of Tuberculosis.

In spite of the development of diagnostic tests for Mycobacterium tuberculosis (M. tuberculosis), the etiological agent of tuberculosis, there has remained a gap between the established methods and an easily accessible diagnostic test, particularly in developing and resource-poor areas. By combining isothermal amplification of IS6110 as the target gene and recognition by DNA-functionalized Au nanoparticles (DNA-AuNPs), we develop a colorimetric LAMP assay for convenient in vitro diagnostics of tuberculosis with a quick (≤50 min) "yes" or "no" readout. The DNA-AuNPs not only tolerate the interference in the complex LAMP system but also afford in situ identification of the amplicon, allowing for colloidal dispersion via steric effect depending on DNA grafting density. The target-induced stabilization and red appearance of the DNA-AuNPs contrast with the occurrence of gray aggregates in a negative sample. Furthermore, the DNA-AuNPs demonstrate excellent performance after long-term (≥7 months) storage while preserving the unsacrificed sensitivity. The high specificity of the DNA-AuNPs is further demonstrated in the naked-eye LAMP assay of M. tuberculosis in patients' sputum samples. Given the rapidity, cost-effectiveness, and instrument-free characteristics, the naked-eye LAMP assay is particularly beneficial for tuberculosis diagnosis in urgent situations and resource-limited settings and can potentially expedite patient care and treatment initiation.

Correction: Effect of DNA density immobilized on gold nanoparticles on nucleic acid detection.

[This corrects the article DOI: 10.1039/D3RA06528F.].

Suppressed DNA base pair stacking assembly of gold nanoparticles in an alcoholic solvent for enhanced ochratoxin A detection in Baijiu.

The currently established DNA nanoprobes for the detection of mycotoxin from beverages have been limited by complicated sample pretreatment and uncontrollable nanoparticle flocculation in complex systems. We develop a rapid colorimetric approach for ochratoxin A (OTA) detection in Baijiu in a sample-in/"yes" or "no" answer-out fashion through target-modulated base pair stacking assembly of DNA-functionalized gold nanoparticles (DNA-AuNPs). The colorimetric signification of OTA relies on the competition of OTA with the AuNP surface-grafted DNA in binding with an OTA-targeted aptamer. The specific recognition of OTA by the aptamer prevents DNA duplex formation on the AuNP surface, thereby inhibiting the base pair stacking assembly of the DNA-AuNPs and giving rise to a "turn-on" color. By further suppressing DNA hybridization using a bulged loop design and an alcohol solution, the DNA-AuNPs exhibit an improved reproducibility for OTA sensing while maintaining excellent susceptivity to OTA. A detection limit of 88 nM was achieved along with high specificity towards OTA, which is lower than the maximum tolerated level of OTA in foodstuffs defined by countries worldwide. The entire reaction time, avoiding sample pretreatment, is less than 17 min. The DNA-AuNPs with anti-interference features and sensitive "turn-on" performance promise convenient on-site detection of mycotoxin from daily beverages.

A Simple Colorimetric Assay of Bleomycin-Mediated DNA Cleavage Utilizing Double-Stranded DNA-Modified Gold Nanoparticles.

A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double-stranded (ds) DNA-modified gold nanoparticles (dsDNA-AuNPs) possessing a cleavage site. The assay was performed using dsDNA-AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10-min exposure in dsDNA-AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt-induced non-crosslinking aggregation of dsDNA-AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA-AuNPs caused by BLM-mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA-cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents.

Design of a Sensitive Extracellular Vesicle Detection Method Utilizing a Surface-Functionalized Power-Free Microchip.

Extracellular vesicles (EVs), which are small membrane vesicles secreted from cells into bodily fluids, are promising candidates as biomarkers for various diseases. We propose a simple, highly sensitive method for detecting EVs using a microchip. The limit of detection (LOD) for EVs was improved 29-fold by changing the microchannel structure of the microchip and by optimizing the EV detection protocols. The height of the microchannel was changed from 25 to 8 µm only at the detection region, and the time for EV capture was extended from 5 to 10 min. The LOD was 6.3 × 1010 particles/mL, which is lower than the concentration of EVs in the blood. The detection time was 19 min, and the volume of EV solution used was 2.0 µL. These results indicate that an efficient supply of EVs to the detection region is effective in improving the sensitivity of EV detection. The proposed EV detection method is expected to contribute to the establishment of EV-based cancer point-of-care testing.

Generation of transmitochondrial cybrids using a microfluidic device.

Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0 cells with puromycin-based selection in the microfluidic device.

G-Quadruplex-Functionalized Gold Nanoparticles for a Real-Time Biomolecule Sensor with On-Demand Tunable Properties.

G-quadruplex (G4) DNA-functionalized gold nanoparticles (AuNPs) were fabricated for a new sensing platform for a biomolecule, thrombin. Thrombin-binding aptamer (TBA), which forms a highly ordered G4 structure, was immobilized on AuNPs. The particles were induced to aggregate by binding of thrombin to G4 DNA. Thrombin was thus detected by the color change of the colloidal system from red to purple-blue. The aggregation was not due to the bridging between the particles through thrombin but to the reduction in steric repulsion attributable to the mobility and flexibility of G4 DNA. The change in the colloidal stability was quick and the bathochromic peak shift varied with the concentration of thrombin. The sensor showed a high specificity to the thrombin target over major proteins in human serum. The detection sensitivity and analytical performance were successfully tuned for an on-demand sensor with a linearity of 10.0-40.0 nM. The limits of detection and of quantification were 3.6 and 10.7 nM, respectively.

Regeneration of the Eyespot and Flagellum in Euglena gracilis during Cell Division.

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26-97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis.

Temporal Evolution of the Gravitaxis of Euglena gracilis from a Single Cell.

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 mm × 2.5 mm × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized.

Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.

Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ0) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.

Plasmon switching of gold nanoparticles through thermo-responsive terminal breathing of surface-grafted DNA in hydrated ionic liquids.

Self-assembly performed in ionic liquids (ILs) as a unique solvent promises distinct functions and applications in sensors, therapeutics, and optoelectronic devices due to the rich interactions between nanoparticle building blocks and ILs. However, the general consideration that common nanoparticles are readily destabilized by counterions in an IL has largely prevented researchers from investigating controlled nanoparticle assembly in IL-based systems. This study explores the assembling behaviour of double-stranded (ds) DNA-functionalized gold nanoparticles (dsDNA-AuNPs) in hydrated ionic liquids. The DNA base pair stacking assembly of dsDNA-AuNPs occurs at a low IL concentration (<5%). However, a moderate ionic liquid concentration (5-40%) can de-hybridize dsDNA and leaves single-stranded (ss) DNA stabilizing the AuNPs. In concentrated ionic liquids (>40%), interestingly, the higher ionic strength leads to the assembly of DNA-AuNPs. The triphasic assembly trend is also generally observed regardless of the type of IL. By down-regulation of DNA's melting temperature with the IL, the assembly of DNA-AuNPs affords robust response to a lower temperature range, promising applications in plasmonic devices and range-tunable temperature sensors.

Terminal Sequence-Specific Interparticle Attraction between DNA Duplex-Carrying Polystyrene Microparticles in Aqueous Salt Solution Assessed by Optical Tweezers.

The dispersion behavior of DNA duplex-carrying colloidal particles in aqueous high-salt solutions shows extraordinary selectivity against the duplex terminal sequence. We investigated the interparticle force between DNA duplex-carrying polystyrene (dsDNA-PS) microparticles in aqueous salt solutions and examined their behavior in relation to the duplex terminal sequences. Force-distance (F-D) curves for a pair of dsDNA-PS particles were recorded with a dual-beam optical tweezers system with the two optically trapped particles closely approaching each other. Interestingly, only 3-5% of the oligo-DNA strands on the dsDNA-PS particles formed a duplex with complementary DNAs, and the F-D curves showed a distinct specificity to the duplex terminal sequences in the interparticle force at a high-NaCl concentration; a clear attraction peak was observed in F-D curves only when the duplex terminal was a complementary base pair. The attractive strength reached 2.6 ± 0.5 pN at 500 mM NaCl and 4.3 ± 1.0 pN at 750 mM NaCl. By sharp contrast, no significant attraction occurred for the particles with mismatched duplex terminals even at 750 mM NaCl. Similar duplex terminal-specificity in the interparticle force was also confirmed for dsDNA-PS particles in divalent MgCl2 solutions. Considering that the duplex terminal sequences on the dsDNA-PS particles showed only a negligible difference in their surface charges under identical salt conditions, we concluded that the interparticle attraction observed only for the dsDNA-PS particles with complementary duplex terminals is attributable to the salt-facilitated stacking interaction between the paired terminal nucleobases (i.e., blunt-end stacking) on the dsDNA-PS surfaces. Our results thus demonstrate the occurrence of a duplex terminal-specific interparticle force between dsDNA-PS particles under high-salt conditions.

Multiplex MicroRNA Detection on a Surface-Functionalized Power-Free Microfluidic Chip.

Circulating microRNAs (miRNAs) have emerged as promising cancer biomarkers because their concentration profiles in body fluids are associated with the type and clinical stage of cancer. For multiplex miRNA detection, a novel surface-functionalized power-free microfluidic chip (SF-PF microchip) has been developed. The inner surface of the SF-PF microchip microchannels was functionalized via electron beam-induced graft polymerization and immobilization of capture probe DNAs. Simultaneous and specific duplex miRNA detection was achieved on the line-type SF-PF microchip with detection limits of 19.1 and 47.6 nmol L-1 for hsa-miR-16 and hsa-miR-500a-3p, respectively. Moreover, simultaneous and specific triplex miRNA detection was achieved on the stripe-type SF-PF microchip. The sample volume required for this microchip was 0.5 µL, and the time required for detection was 17 min. These results indicate that up to six types of miRNAs could be detected without compromising the advantages of the previous SF-PF microchips for cancer point-of-care testing.

Electrochemical Impedimetric Study of Non-Watson-Crick Base Pairs of DNA.

Electrochemical impedance spectroscopy (EIS) was used to detect non-Watson-Crick base pairs of DNA. Thiol-modified DNA as a probe and mercaptohexanol (MCH) were co-immobilized to form a DNA/MCH mixed self-assembled monolayer on a gold electrode surface and then hybridized with complementary DNAs. The DNA layers were measured by the EIS method and interpreted by equivalent circuits. Every terminal base mismatch of the DNA duplex brought about an increase in the charge-transfer resistance (Rct), unlike the case with a fully matched DNA duplex. The value of Rct was highly sensitive to the number of base mismatches for both unpaired and overhang DNA at the terminal. For internal base mismatches, however, no significant increase in Rct was observed. These experimental results proved that the charge transfer of redox molecules to the electrode surface is largely hindered by an end fraying motion due to base unpairing and dangling overhang. EIS was able to detect these steric properties of DNA strands. Furthermore, an electrode modified with G-quadruplex (G4) DNA demonstrated the influences of bulkiness and loop structure on the accessibility of the redox probe to the electrode.

DNA Terminal-Specific Dispersion Behavior of Polystyrene Latex Microparticles Densely Covered with Oligo-DNA Strands Under High-Salt Conditions.

We prepared microspheres densely covered with oligo-DNA strands by immobilizing amino-terminated oligo-DNA strands on the surface of carboxylate polystyrene latex (PS) particles via the amide bond formation. The obtained microspheres (ssDNA-PS) stably dispersed in neutral pH buffer containing high concentrations of NaCl. For the ssDNA-PS ≥1 µm diameter, only 3 - 5% of surface-immobilized oligo-DNA could form a duplex with the complementary strands. Nevertheless, the resulting ssDNA-PS showed a distinct duplex terminal dependency in their dispersion behavior under neutral pH and high NaCl conditions; the microspheres with fully-matched duplexes on the surface spontaneously aggregated in a non-crosslinking manner. By contrast, the microspheres with terminal-mismatched duplexes remained dispersed under the identical conditions. These results suggest that the micrometer-scale particles covered with oligo-DNA strands also have high susceptibility to a duplex terminal sequence in their dispersion property, similar to previously reported DNA-functionalized nanoparticles. This property could potentially be used in various applications including analytical purposes.

A Microfluidic Device for Modulation of Organellar Heterogeneity in Live Single Cells.

The quantitatively controlled organellar transfer between living single cells provides a unique experimental platform to analyze the contribution of organellar heterogeneity on cellular phenotypes. We previously developed a microfluidic device which can perform quantitatively controlled mitochondrial transfer between live single cells by promoting strictured cytoplasmic connections between live single cells, but its application to other organelles is unclear. In this study, we investigated the quantitative properties of peroxisome transfer in our microfluidic device. When cells were fused through a 10 or 4 µm long microtunnel by a Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel, and a subsequent recovery culture disconnected the fused cells. The peroxisome number being transferred through a 10 µm length of the microtunnel was smaller than that of 4 µm. These data suggest that our microfuidic device can perform a quantitative control of peroxisome transfer.

Protein-Functionalized Gold Nanoparticles for Antibody Detection Using the Darkfield Microscopic Observation of Nanoparticle Aggregation.

Gold nanoparticles (AuNPs) are commonly used in biosensing applications. In this study, AuNPs were synthesized by using reduced bovine serum albumin (rBSA) as the reducing agent. The rBSA conjugated with AuNPs via Au-Sulfur interactions to form rBSA-functionalized AuNPs (rBSA-AuNPs). The interaction of the rBSA moieties on the rBSA-AuNP surface with an anti-BSA antibody (anti-BSA) led to AuNP aggregation, which enabled the successful detection of anti-BSA at a concentration as low as 20 nM through darkfield microscopy (DFM). This study demonstrates the potential applications of protein-functionalized AuNPs in the bioanalysis of substances through DFM.

DNA Base Pair Stacking Assembly of Anisotropic Nanoparticles for Biosensing and Ordered Assembly.

Anisotropic gold nanoparticles have attracted great interest due to their unique physicochemical properties derived from the shape anisotropy. Manipulation of their interfacial interactions, and thereby the assembling behaviors are often requisite in their applications ranging from optical sensing and diagnosis to self-assembly. Recently, the control of interfacial force based on base pair stacking of DNA terminals have offered a new avenue to surface engineering of nanostructures. In this review, we focus on the DNA base stacking-induced assembly of anisotropic gold nanoparticles, such as nanorods and nanotriangles. The fundamental aspects of anisotropic gold nanoparticles are provided, including the mechanism of the anisotropic growth, the properties arising from the anisotropic shape, and the construction of DNA-grafted anisotropic gold nanoparticles. Then, the advanced applications of their functional assemblies in biosensing and ordered assembly are summarized, followed by a comparison with gold nanospheres. Finally, conclusions and the direction of outlooks are given including future challenges and opportunities in this field.

Molecular detection using aptamer-modified gold nanoparticles with an immobilized DNA brush for the prevention of non-specific aggregation.

Gold nanoparticles (AuNPs) are often used for biosensing. In particular, aptamer-modified AuNPs are often used for colorimetric molecular detection, where target molecule-induced AuNP aggregates can be recognized by a color change from red to blue. However, non-specific aggregation could be induced by various compounds, leading to false-positive results. In this work we employed high-density ssDNA modification on the AuNP surface to prevent non-specific aggregation. The covalently immobilized DNA brush was used as an anchor for an aptamer specific for the target molecule. Herein, as a proof-of-concept study, we demonstrated detection of estradiol (E2), one of the endocrine-disrupting estrogen molecules as a model target, in the presence of antibiotic kanamycin (KN) as a model of co-contaminating compounds that induce non-specific aggregation of AuNPs. We also developed a smartphone dark field microscope (DFM) to visualize AuNP aggregation. Our previous study demonstrated that the observation of light scattering by AuNP aggregates with DFM can be applied for versatile molecular detection. In this work, we could successfully detect E2 with the smartphone DFM, and the results were verified by the results from a conventional benchtop DFM. This study would contribute to the future field applicability of AuNP-based sensors.