Potentiometric Multichannel Cytometer Microchip for High-throughput Microdispersion Analysis

The parallelization of microfluidic cytometry is expected to lead to considerably enhanced throughput enabling point-of-care diagnosis. In this article, the development of a microfluidic potentiometric multichannel cytometer is presented. Parallelized microfluidic channels sharing a fluid path inevitably suffer from interchannel signal crosstalk that results from electrical coupling within the microfluidic channel network. By employing three planar electrodes within a single detection channel, we electrically decoupled each channel unit, thereby enabling parallel analysis by using a single cytometer microchip with multiple microfluidic channels. The triple-electrode configuration is validated by analyzing the size and concentration of polystyrene microbeads (diameters: 1.99, 2.58, 3, and 3.68 μm; concentration range: ∼2 × 10⁵ mL^–1 to ∼1 × 10⁷ mL^–1) and bacterial microdispersion samples (<i>Bacillus subtilis</i>, concentration range: ∼4 × 10⁵ CFU mL^–1 to ∼3 × 10⁶ CFU mL^–1). Crosstalk-free parallelized analysis is then demonstrated using a 16-channel potentiometric cytometer (maximum cross-correlation coefficients |<i>r</i>|: < 0.13 in all channel combinations). A detection throughput of ∼48 000 s^–1 was achieved; the throughout can be easily increased with the degree of parallelism of a single microchip without additional technical complexities. Therefore, this methodology should enable high-throughput and low-cost cytometry

Tuning and Maximizing the Single-Molecule Surface-Enhanced Raman Scattering from DNA-Tethered Nanodumbbells

We extensively study the relationships between single-molecule surface-enhanced Raman scattering (SMSERS) intensity, enhancement factor (EF) distribution over many particles, interparticle distance, particle size/shape/composition and excitation laser wavelength using the single-particle AFM-correlated Raman measurement method and theoretical calculations. Two different single-DNA-tethered Au–Ag core–shell nanodumbbell (GSND) designs with an engineerable nanogap were used in this study: the GSND-I with various interparticle nanogaps from ∼4.8 nm to <1 nm or with no gap and the GSND-II with the fixed interparticle gap size and varying particle size from a 23–30 nm pair to a 50–60 nm pair. From the GSND-I, we learned that synthesizing a <1 nm gap is a key to obtain strong SMSERS signals with a narrow EF value distribution. Importantly, in the case of the GSND-I with <1 nm interparticle gap, an EF value of as high as 5.9 × 10¹³ (average value = 1.8 × 10¹³) was obtained and the EF values of analyzed particles were narrowly distributed between 1.9 × 10¹² and 5.9 × 10¹³. In the case of the GSND-II probes, a combination of >50 nm Au cores and 514.5 nm laser wavelength that matches well with Ag shell generated stronger SMSERS signals with a more narrow EF distribution than <50 nm Au cores with 514.5 nm laser or the GSND-II structures with 632.8 nm laser. Our results show the usefulness and flexibility of these GSND structures in studying and obtaining SMSERS structures with a narrow distribution of high EF values and that the GSNDs with < 1 nm are promising SERS probes with highly sensitive and quantitative detection capability when optimally designed

Accurate Diagnosis of COVID-19 from Self-Collectable Biospecimens Using Synthetic Apolipoprotein H Peptide-Coated Nanoparticle Assay

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses

Workflow of filtering variants for the detection of candidate mutations.

<p>Flowchart shows the pipeline we used for filtering variants. Following exclusion of low quality variants (<20x total reads or <10 allele counts), synonymous, noncoding variants and polymorphisms were discarded. When recurrently reported in COSMIC V60 database, the variants were rescued.</p

Dysplastic eosinophils frequently observed in IHE/IHES patients harboring mutations (n = 7).

<p>Cytoplasms are filled with abnormal secondary basophilic granules (BM, Wright-Giemsa, 1000×). Dysplastic eosinophils were more common in the mutation-positive group than in the mutation-negative group (<i>P</i> = 0.045).</p

An illustration indicating action levels of discovered genes in the present study in relation to eosinophil production and Pathway Studio network analysis.

<p>Networks were created based on at least one published reference regarding candidate genes and the known 14 genes related to eosinophil production. (A) Genes marked with blue letter are well-known genes for which mechanisms are proven in eosinophil production. (B) Genes exerting on eosinophil lineage commitment at hematopoietic stem cell level. (C) Genes exerting at eosinophil lineage commitment and prolongation of eosinophil survival. (D) Eosinophil recruitment into tissue. (E) Genes interacting with IL-5, pivotal to eosinophil production and differentiation. <i>GATA1</i> and <i>CEBPA</i> were excluded from the network because they are involved in both eosinophil lineage commitment and the candidate gene set.</p

Distribution of mutated genes in idiopathic hypereosinophilia patients.

<p>The frequency of candidate mutations in each gene was listed for the all 16 patients with mutation positive.</p

Correlations between frequently mutated genes (more than 2 patients).

<p>Statistically significant correlations (<i>P</i> < 0.05) were indicated. The correlation coefficients are shown by a color gradient and size difference.</p

Mutated genes in patients with eosinophilia (n = 16).

<p>Mutated genes in patients with eosinophilia (n = 16).</p

Patient clinical characteristics according to somatic mutation status.

<p>Patient clinical characteristics according to somatic mutation status.</p