Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer.

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 µL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.

Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification.

We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 µL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.

A competitive immunoassay system for microfluidic paper-based analytical detection of small size molecules.

The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (µPADs) is reported. The µPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. µPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the µPAD detection of biotin as a model compound with a detection limit of 0.10 µg mL-1. The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B1, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL-1. The µPAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.

Image analysis for a microfluidic paper-based analytical device using the CIE L*a*b* color system.

The combination of a microfluidic paper-based analytical device (µPAD) and digital image analysis is widely used for quantitative analysis with µPADs because of its easy and simple operation. Herein, we have demonstrated a quantitative analysis based on multiple color changes on a µPAD. The CIE L*a*b* color system was employed to analyse the digital images obtained with the µPAD. We made pH measurements using a universal pH-indicator showing multiple color changes for various pH values of aqueous test solutions. The detectable pH range of this method was wider than the typical grayscale-based image analysis, and we succeeded in the measurements for a wide pH range of 2-9.

Novel concept of washing for microfluidic paper-based analytical devices based on capillary force of paper substrates.

A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow µPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model µPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 µg mL-1. Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported.

Differences between cardiac memory T wave changes after idiopathic left ventricular tachycardia and ischemic T wave inversion induced by acute coronary syndrome.

BACKGROUND: Cardiac memory (CM) after idiopathic left ventricular tachycardia (ILVT) mimics ischemic T wave inversion (TWI) induced by acute coronary syndrome (ACS). We aimed to establish electrocardiography criteria for differentiating the CM from ischemic TWI. METHODS AND RESULTS: We evaluated 16 ILVT and 48 ACS patients. We identified TWI after ILVT in 9/16 patients (CM group), typically in leads II, III, aVF, aVR, and V4-6. The characteristics of CM were similar to TWI induced by ACS involving right coronary artery, but the CM group had more TWI in V4 and shorter QTc. The criteria of (1) positive T in aVL, (2) negative or isoelectric T in II, and (3) negative T in V4-6 or (4) QTc <430ms were 100% sensitive and 96% specific for the CM group. CONCLUSION: CM after ILVT can be differentiated in most cases from ischemic TWI by the distribution of TWI and the QTc.

Fluorescence polarization measurement system using a liquid crystal layer and an image sensor.

The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.

An instrument-free, screen-printed paper microfluidic device that enables bio and chemical sensing.

This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 µm, as obtained by this method. Fabricated microfluidic paper-based analytical devices (µPADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 µM, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for µPADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.

Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations.

Lipid nanoparticles (LNPs) are essential carrier particles in drug delivery systems, particularly in ribonucleic acid delivery. In preparing lipid-based nanoparticles, microfluidic-based ethanol injection may produce precisely size-controlled nanoparticles. Ethanol is critical in LNP formation and post-treatment processes and affects liposome size, structure, lamellarity, and drug-loading efficiency. However, the effects of time-dependent changes in the ethanol concentration on the structural dynamics of liposomes are not clearly understood. Herein, we investigated ethanol-induced lipid bilayer changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations. The time-resolved SAXS measurement system revealed that single unilamellar liposomes were converted to multilamellar liposomes within 0.8 s of contact with ethanol, and the d-spacing was decreased from 6.1 (w/o ethanol) to 4.4 nm (80% ethanol) with increasing ethanol concentration. We conducted 1 µs MD simulations to understand the molecular-level structural changes in the liposomes. The MD simulations revealed that the changes in the lamellar structure caused by ethanol at the molecular level could explain the structural changes in the liposomes observed via time-resolved SAXS. Therefore, the post-treatment process to remove residual ethanol is critical in liposome formation.

A case of coronary artery compression syndrome resulting from peri-valvular regurgitation and long-standing atrial fibrillation.

A man in his 70s with a history of mitral valve replacement (MVR) and long-standing persistent atrial fibrillation (AF) presented with effort angina. Coronary angiography revealed severe stenosis of the left main coronary artery (LMCA). As it was an emergent case, PCI (percutaneous coronary intervention) was selected for treatment. Intravascular ultrasonography revealed no atherosclerotic lesions in the LMCA. The LMCA was effectively dilated by the drug-eluting stent. No elevation in intracardiac pressure was observed in cardiac catheterization after PCI. Computed tomography scan indicated potential compression of the LMCA by the surrounding structures. In cases of long-standing persistent AF and an enlarged atrium after MVR, the possibility of LMCA stenosis due to anatomical changes should be considered. Learning Objectives: ◾Peri-valvular regurgitation and long-standing persistent atrial fibrillation can potentially cause atrial enlargement.◾Coronary artery stenosis without atherosclerosis can occur due to compression from surrounding structures or shifting of the coronary artery.◾Stent therapy provides a temporary solution and coronary artery bypass grafting or switching should be considered if re-stenosis occurs.

Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.

RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.

A portable liquid chromatography system based on a separation/detection chip module consisting of a replaceable ultraviolet-visible absorbance or contactless conductivity detection unit.

Recently, there has been a growing demand for miniaturized analytical instruments, including portable HPLC systems, that can enable rapid analysis in the field. This study aimed to develop chip-based separation/detection modules with replaceable detection units for constructing compact HPLC systems to minimize the dead volume. This module provides a tubing-free connection between the column and the detection unit. This study also built detection units for conductivity detection and ultraviolet-visible (UV-Vis) detection to cover a wide variety of inorganic and organic compounds. Furthermore, UV- and Vis-light-emitting diodes were employed for the absorbance detection unit. In addition, portable all-in-one HPLC systems and a handy HPLC system were constructed for ion chromatography and reversed-phase chromatography, demonstrating the successful separation and detection of inorganic ions and several organic compounds.

A case of retractable helix lead auto-retraction: A possible cause of deep septal lead dislodgement.

This paper explains the phenomenon where the helix lead automatically retracts because of residual torque during deep septal pacing.

Clinical impact of optical coherence tomography findings after drug-coated balloon treatment for patients with acute coronary syndromes.

BACKGROUND: Drug-coated balloon (DCB) became a potential treatment option for patients with acute coronary syndrome (ACS); however, factors associated with target lesion failure (TLF) remain uncertain. METHODS: This retrospective, multicentre, observational study included consecutive ACS patients who underwent optical coherence tomography (OCT)-guided DCB treatment. Patients were divided into two groups according to the occurrence of TLF, a composite of cardiac death, target vessel-related myocardial infarction, and ischemia-driven target lesion revascularisation. RESULTS: We enrolled 127 patients in this study. During the median follow-up period of 562 (IQR: 342-1164) days, 24 patients (18.9%) experienced TLF, and 103 patients (81.1%) did not. The cumulative 3-year incidence of TLF was 22.0%. The cumulative 3-year incidence of TLF was the lowest in patients with plaque erosion (PE) (7.5%), followed by those with rupture (PR) (26.1%) and calcified nodule (CN) (43.5%). Multivariable Cox regression analysis revealed that plaque morphology was independently associated with TLF on pre-PCI (percutaneous coronary intervention) OCT, and residual thrombus burden (TB) was positively associated with TLF on post-PCI OCT. Further stratification by post-PCI TB revealed a comparable incidence of TLF in patients with PR (4.2%) to that of PE if the culprit lesion had a smaller post-PCI TB than the cut-off value (8.4%). TLF incidence was high in patients with CN, regardless of TB size on post-PCI OCT. CONCLUSIONS: Plaque morphology was strongly associated with TLF for ACS patients after DCB treatment. Residual TB post-PCI might be a key determinant for TLF, especially in patients with PR.

Reentrant ventricular outflow tract tachycardia arising from focal scar detected by delayed enhancement magnetic resonance imaging.

A 58-year-old man was referred to our emergency room with hemodynamically unstable sustained ventricular tachycardia (VT). The morphology of the VT exhibited a left bundle branch block and inferior axis deviation. He had no past history of cardiovascular disease. Echocardiography, cardiac catheterization, cardiac biopsy, gallium scintigram, myocardial scintigram, T1,T2-weighted magnetic resonance imaging (MRI), and gadolinium-enhanced cine MRI did not detect any structural heart disease or abnormal cardiac function. However, delayed-enhancement MRI (DE-MRI) detected a focal intramural scar within the septal ventricular outflow tract. An electrophysiological study revealed a sustained VT with several morphologies and the entrainment phenomenon. Radiofrequency catheter ablation to the site corresponding to the focal scar detected by DE-MRI successfully eliminated the VT.

Impact of post physiological assessment after treatment for de novo coronary lesions using drug-coated balloons.

BACKGROUND: Acute physiological changes after balloon angioplasty are very important because of acute recoil and dissection. However, serial physiological assessments after drug-coated balloons (DCB) have not been investigated. METHODS: This prospective observational single-center study evaluated 50 lesions that underwent optical coherence tomography (OCT)-guided percutaneous coronary intervention (PCI) with DCB and a 9-months angiographical and OCT follow-up. Fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) were measured immediately (FFR0m and, iFR0m) and 15 min (FFR15m and iFR15m) after DCB, and the difference (dif-FFR and, dif-iFR) was calculated. The iFR gradients during lesions treated with DCB were measured. For OCT and quantitative coronary angiography (QCA) data, delta values were calculated. RESULTS: At index PCI, three lesions were needed for bailout stenting. At follow-up, 47 lesions were divided into two groups according to the delta minimal lumen area (MLA) on OCT: late lumen enlargement (LLE) (n = 29) and non-LLE (n = 18). In LLE group, FFR15m and iFR15m were significantly high (0.90 ± 0.03 vs. 0.85 ± 0.07, p < 0.001, 0.97 ± 0.02 vs. 0.92 ± 0.10, p = 0.008, respectively) and %AS on OCT, dif-FFR and dif-iFR were significantly low (38.5% (33.6, 42.3) vs. 45.1% (38.9, 54.6), p = 0.03, -0.001 ± 0.006 vs. 0.036 ± 0.032, p < 0.001, -0.002 ± 0.008 vs. 0.019 ± 0.017, p < 0.001, respectively) compared with non-LLE group. However, there were no significant differences in FFR0m, iFR0m, or any other OCT or QCA data. CONCLUSIONS: Post-physiological assessment and a decrease in physiological indices in the first 15 min after PCI are important for treating de novo lesions using the DCB strategy.

Preparation of size-tunable sub-200 nm PLGA-based nanoparticles with a wide size range using a microfluidic platform.

The realization of poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) from laboratory to clinical applications remains slow, partly because of the lack of precise control of each condition in the preparation process and the rich selectivity of nanoparticles with diverse characteristics. Employing PLGA NPs to establish a large range of size-controlled drug delivery systems and achieve size-selective drug delivery targeting remains a challenge for therapeutic development for different diseases. In this study, we employed a microfluidic device to control the size of PLGA NPs. PLGA, poly (ethylene glycol)-methyl ether block poly (lactic-co-glycolide) (PEG-PLGA), and blend (PLGA + PEG-PLGA) NPs were engineered with defined sizes. Blend NPs exhibit the widest size range (40-114 nm) by simply changing the flow rate conditions without changing the precursor (polymer molecular weight, concentration, and chain segment composition). A model hydrophobic drug, paclitaxel (PTX), was encapsulated in the NPs, and the PTX-loaded NPs maintained a large range of controllable NP sizes. Furthermore, size-controlled NPs were used to investigate the effect of particle size of sub-200 nm NPs on tumor cell growth. The 52 nm NPs showed higher cell growth inhibition than 109 nm NPs. Our method allows the preparation of biodegradable NPs with a large size range without changing polymer precursors as well as the nondemanding fluid conditions. In addition, our model can be applied to elucidate the role of particle sizes of sub-200 nm particles in various biomedical applications, which may help develop suitable drugs for different diseases.

Effect of Organic Solvents on a Production of PLGA-Based Drug-Loaded Nanoparticles Using a Microfluidic Device.

The translation of nanoparticles (NPs) from laboratory to clinical settings is limited, which is not ideal. One of the reasons for this is that we currently have limited ability to precisely regulate various physicochemical parameters of nanoparticles. This has made it difficult to rapidly perform targeted screening of drug preparation conditions. In this study, we attempted to broaden the range of preparation conditions for particle size-modulated poly(lactic-co-glycolic-acid) (PLGA) NP to enhance their applicability for drug delivery systems (DDS). This was done using a variety of organic solvents and a glass-based microfluidic device. Furthermore, we compared the PDMS-based microfluidic device to the glass-based microfluidic device in terms of the possibility of a wider range of preparation conditions, especially the effect of different solvents on the size of the PLGA NPs. PLGA NPs with different sizes (sub-200 nm) were successfully prepared, and three different types of taxanes were employed for encapsulation. The drug-loaded NPs showed size-dependent cytotoxicity in cellular assays, regardless of the taxane drug used.