Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

BACKGROUND: Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS: The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS: Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION: Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Surface Adaptable and Adhesion Controllable Dry Adhesive with Shape Memory Polymer.

Gecko feet consist of numerous micro/nano hierarchical hairs and exhibit a high adhesion onto various surfaces by van der Waals forces. The gecko, despite its mighty adhesion, can travel efficiently with a rapid adhesion switching due to the end of the hairs on the gecko feet are slanted in one direction. Herein, a shape memory polymer (SMP)-based switchable dry adhesive (SSA), inspired by gecko feet, having tremendous surface adaptability and adhesion switching capability, is reported. The SSA shows not only high adhesion to the various surfaces (≈332.8 kPa) but also easy detachment (nearly 3.73 kPa) due to the characteristic of SMP, which can reversibly recover from a deformed shape to its initial shape. On the basis of the novel adhesion switching property, it is suggested the SSA-applied advanced glass transfer system can lead to feasible application. This experiment confirms that an ultrathin and light glass film is transferred easily and sustainably, and it is believed that the SSA may be a breakthrough and a powerful alternative for not only conventional dry adhesives but also the next-level transfer systems.

Inhibitory Effect of Elaeagnus umbellata Fractions on Melanogenesis in α-MSH-Stimulated B16-F10 Melanoma Cells.

When skin is exposed to UV radiation, melanocytes produce melanin. Excessive melanin production leads to skin pigmentation, which causes various cosmetic and health problems. Therefore, the development of safe, natural therapeutics that inhibit the production of melanin is necessary. Elaeagnus umbellata (EU) has long been widely used as a folk medicinal plant because of pharmacological properties that include anti-ulcer, antioxidant, and anti-inflammatory properties. In this study, we investigated the antioxidant activity and melanogenesis inhibitory effects of EU fractions in B16-F10 melanoma cells. EU fractions showed a dose-dependent increase in antioxidant activity in radical scavenging activity. In addition, we evaluated the effect of EU fractions on tyrosinase activity and melanogenesis in α-melanocyte-stimulating hormone-induced B16-F10 melanoma cells. EU was noncytotoxic at 12.5-50 µg/mL. EU fractions effectively inhibited tyrosinase activity and melanogenesis, suppressed the phosphorylation of CREB and ERK involved in the melanogenesis pathway, and down-regulated expression of melanogenesis-related proteins. Interestingly, the anti-melanogenesis effect was most effective at a concentration of 50 µg/mL EU, and the effects of the fractions were superior to those of the extract. Therefore, our study suggests that EU has potential as a safe treatment for excessive pigmentation or as a natural ingredient in cosmetics.

Newborn screening by matrix-assisted laser desorption/ionization mass spectrometry based on parylene-matrix chip.

Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z < 500). Reproducibility of inter-spot and intra-spot analyses of amino acids was less than 10%. Methanol extraction was adopted for simple and rapid sample preparation of serum before mass spectrometric analysis showing 13.3 to 45% of extraction efficiency. Calibration curves for diagnosis of neonatal metabolic disorders were obtained by analyzing methanol-extracted serum spiked with target amino acids using MALDI-ToF MS. They showed good linearity (R2 > 0.98) and the LODs were ranging from 9.0 to 22.9 µg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders.

Organic Mixed Ionic-Electronic Conductors for Bioelectronic Sensors: Materials and Operation Mechanisms.

The field of organic mixed ionic-electronic conductors (OMIECs) has gained significant attention due to their ability to transport both electrons and ions, making them promising candidates for various applications. Initially focused on inorganic materials, the exploration of mixed conduction has expanded to organic materials, especially polymers, owing to their advantages such as solution processability, flexibility, and property tunability. OMIECs, particularly in the form of polymers, possess both electronic and ionic transport functionalities. This review provides an overview of OMIECs in various aspects covering mechanisms of charge transport including electronic transport, ionic transport, and ionic-electronic coupling, as well as conducting/semiconducting conjugated polymers and their applications in organic bioelectronics, including (multi)sensors, neuromorphic devices, and electrochromic devices. OMIECs show promise in organic bioelectronics due to their compatibility with biological systems and the ability to modulate electronic conduction and ionic transport, resembling the principles of biological systems. Organic electrochemical transistors (OECTs) based on OMIECs offer significant potential for bioelectronic applications, responding to external stimuli through modulation of ionic transport. An in-depth review of recent research achievements in organic bioelectronic applications using OMIECs, categorized based on physical and chemical stimuli as well as neuromorphic devices and circuit applications, is presented.

A pattern recognition artificial olfactory system based on human olfactory receptors and organic synaptic devices.

Neuromorphic sensors, designed to emulate natural sensory systems, hold the promise of revolutionizing data extraction by facilitating rapid and energy-efficient analysis of extensive datasets. However, a challenge lies in accurately distinguishing specific analytes within mixtures of chemically similar compounds using existing neuromorphic chemical sensors. In this study, we present an artificial olfactory system (AOS), developed through the integration of human olfactory receptors (hORs) and artificial synapses. This AOS is engineered by interfacing an hOR-functionalized extended gate with an organic synaptic device. The AOS generates distinct patterns for odorants and mixtures thereof, at the molecular chain length level, attributed to specific hOR-odorant binding affinities. This approach enables precise pattern recognition via training and inference simulations. These findings establish a foundation for the development of high-performance sensor platforms and artificial sensory systems, which are ideal for applications in wearable and implantable devices.

Plasma Deposition of Parylene-like Films with Chemical Functional Groups for Immunoassays.

Parylene-like films obtained via the plasma decomposition of parylene precursors with functional groups (amino and formyl) are proposed as an alternative to those obtained via the thermal method. To analyze the chemical functional groups after plasma deposition, a surface analysis of the parylene films using the two different deposition methods was performed via Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS). The FT-IR analysis revealed that the featured peaks of the chemical functional groups were maintained in the parylene-like films obtained via the plasma deposition method. The XPS analysis revealed that the featured chemical functional groups of parylene-AM and parylene-H were maintained after plasma deposition. The surface energy of the parylene films was estimated by using contact angle measurements. The plasma-deposited parylene films were then employed for protein immobilization via the functional groups using horseradish peroxidase (44 kDa) and green fluorescent protein (25 kDa) as model proteins. The parylene-AM and parylene-H films obtained via plasma deposition exhibited higher immobilization efficiencies than did the same parylene films obtained via thermal deposition. Finally, a competitive immunoassay was obtained by immobilizing the Fv-antibodies on plasma-deposited parylene-AM and parylene-H films via covalent bonding. Using heat-deactivated SARS-CoV-2 as a real sample, the limit of detection at the feasible level for the medical diagnosis of COVID-19 was achieved using a competitive immunoassay based on immobilized Fv-antibodies on plasma-deposited parylene films.

Wide tuning range wavelength-swept laser with a single SOA at 1020 nm for ultrahigh resolution Fourier-domain optical coherence tomography.

In this study, we demonstrated a wide tuning range wavelength-swept laser with a single semiconductor optical amplifier (SOA) at 1020 nm for ultrahigh resolution, Fourier-domain optical coherence tomography (UHR, FD-OCT). The wavelength-swept laser was constructed with an external line-cavity based on a Littman configuration. An optical wavelength selection filter consisted of a grating, a telescope, and a polygon scanner. Before constructing the optical wavelength selection filter, we observed that the optical power, the spectrum bandwidth, and the center wavelength of the SOA were affected by the temperature of the thermoelectric (TE) cooler in the SOA mount as well as the applied current. Therefore, to obtain a wide wavelength tuning range, we adjusted the temperature of the TE cooler in the SOA mount. When the temperature in the TE cooler was 9 °C, our swept source had a tuning range of 142 nm and a full-width at half-maximum (FWHM) of 121.5 nm at 18 kHz. The measured instantaneous spectral bandwidth (δλ) is 0.085 nm, which was measured by an optical spectrum analyzer with a resolution bandwidth of 0.06 nm. This value corresponds to an imaging depth of 3.1 mm in air. Additionally, the averaged optical power of our swept source was 8.2 mW. In UHR, FD/SS-OCT using our swept laser, the measured axial resolution was 4.0 µm in air corresponding to 2.9 µm in tissue (n = 1.35). The sensitivity was measured to be 93.1 dB at a depth of 100 µm. Finally, we obtained retinal images (macular and optic disk) and a corneal image.

Microfabrication of SiN membrane nanosieve using anisotropic reactive ion etching (ARIE) with an Ar/CF4 gas flow.

We have designed, fabricated, and characterized a low-stressed silicon nitride (SiN) membrane nanosieve (100 microm x 100 microm) using an anisotropic reactive ion etching (ARIE) combining with gas mixture, thus maintaining compatibility with the complementary metal-oxide semiconductor integrated circuit (CMOS IC) processes. The holes pattern of this nanosieve membrane was precisely controlled under 30 nm diameter by the electron beam writing. By employing mainly anisotropic reactive ion etching plus diffusion to the depth direction, the etch holes size was controlled to be the same with patterns on the e-beam resist (ER). This nanosieve membrane has proper mechanical strength withstanding up to one bar of transmembrane pressure. And it can endure harsh treatments such as high temperature up to 800 degrees C. In addition, it is inert to a number of strong chemicals including the piranha (H2SO4 + H2O2) solution, highly-concentrated potassium hydroxide (KOH), hydrogen fluoride (HF), hydrogen chloride (HCI), and nitric acid (HNO3).

Gold Nanoparticle-Enhanced and Roll-to-Roll Nanoimprinted LSPR Platform for Detecting Interleukin-10.

Localized surface plasmon resonance (LSPR) is a powerful platform for detecting biomolecules including proteins, nucleotides, and vesicles. Here, we report a colloidal gold (Au) nanoparticle-based assay that enhances the LSPR signal of nanoimprinted Au strips. The binding of the colloidal Au nanoparticle on the Au strip causes a red-shift of the LSPR extinction peak, enabling the detection of interleukin-10 (IL-10) cytokine. For LSPR sensor fabrication, we employed a roll-to-roll nanoimprinting process to create nanograting structures on polyethylene terephthalate (PET) film. By the angled deposition of Au on the PET film, we demonstrated a double-bent Au structure with a strong LSPR extinction peak at ~760 nm. Using the Au LSPR sensor, we developed an enzyme-linked immunosorbent assay (ELISA) protocol by forming a sandwich structure of IL-10 capture antibody/IL-10/IL-10 detection antibody. To enhance the LSPR signal, we introduced colloidal Au nanocube (AuNC) to be cross-linked with IL-10 detection antibody for immunogold assay. Using IL-10 as a model protein, we successfully achieved nanomolar sensitivity. We confirmed that the shift of the extinction peak was improved by 450% due to plasmon coupling between AuNC and Au strip. We expect that the AuNC-assisted LSPR sensor platform can be utilized as a diagnostic tool by providing convenient and fast detection of the LSPR signal.

Remora-Inspired Reversible Adhesive for Underwater Applications.

The remoras are marine species that can effectively move by clinging onto other marine species via a suction disk on their dorsal side, which is composed of complex structures. The inner suction disk could be divided into three large parts, namely, lip, lamella, and spinule. The lamella is deformed actively to generate pressure difference between the inside and outside of the suction disk, and the lip maintains the sealing. The spinule, which is composed of hairs with diameter of 300 µm or less hair, enhances the frictional force. In this study, we easily fabricated polymer-based adhesive inspired by the suction disk of the remora and conducted an experiment to determine its performance. The adhesive exhibited admirable performance with a 26.68 N cm-2 (266.8 kPa) pull-off strength and a 19.42 N cm-2 (194.2 kPa) shear strength in water. The durability test result indicated that the adhesion and friction characteristics of the adhesive were well maintained even after multiple uses.

Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi.

α2,3-Sialyltransferase from Pasteurella multocida (PmST1) is an enzyme that transfers a sialyl group of donor substrates to an acceptor substrate called N-acetyl-d-lactosamine (LacNAc). In this study PmST1 was expressed on the outer membrane of wildtype Escherichia coli (BL21) with lipopolysaccharide (LPS) and ClearColi with no LPS, and then the enzyme activity and expression level of PmST1 were compared. As the first step, the expression levels of PmST1 on the outer membranes of wildtype E. coli (BL21) and ClearColi were compared according to the IPTG induction time, and the absolute amount of surface-displayed PmST1 was calculated using densitometry of SDS-PAGE. As the next step, the influence of LPS on the PmST1 activity was estimated by analyzing Michaelis-Menten plot. The enzyme activity of PmST1 was analyzed by measuring the concentration of CMP, which was a by-product after the transfer of the sialyl group of donor compounds to the acceptor compounds. From a Michaelis-Menten plot, the enzyme activity of the surface-displayed PmST1 and the maximum rate (Vmax) of ClearColi were higher than those of wildtype E. coli (BL21). However, the KM value, which represented the concentration of substrate to reach half the maximum rate (Vmax), was similar for both enzymes. These results represented such a difference in enzyme activity was occurred from the interference of LPS on the mass transport of the donor and acceptor to PmST1 for the sialyl group transfer.

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity.

Directionally Aligned Amorphous Polymer Chains via Electrohydrodynamic-Jet Printing: Analysis of Morphology and Polymer Field-Effect Transistor Characteristics.

Electrohydrodynamic-jet (EHD-jet) printing provides an opportunity to directly assembled amorphous polymer chains in the printed pattern. Herein, an EHD-jet printed amorphous polymer was employed as the active layer for fabrication of organic field-effect transistors (OFETs). Under optimized conditions, the field-effect mobility (µFET) of the EHD-jet printed OFETs was 5 times higher than the highest µFET observed in the spin-coated OFETs, and this improvement was achieved without the use of complex surface templating or additional pre- or post-deposition processing. As the chain alignment can be affected by the surface energy of the dielectric layer in EHD-jet printed OFETs, dielectric layers with varying wettability were examined. Near-edge X-ray absorption fine structure measurements were performed to compare the amorphous chain alignment in OFET active layers prepared by EHD-jet printing and spin coating.

Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren's syndrome.

The objective of this study was to present an immunoassay for the diagnosis of Sjögren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pI<7) on the outer membrane of intact E. coli and LPS-free E. coli resulted in a slight change in zeta potential values. E. coli with autodisplayed La/SSB protein was used for an immunoassay of anti-La/SSB antibodies for the diagnosis of Sjögren's syndrome. The surface of E. coli with the autodisplayed antigen was modified with rabbit serum and papain to prevent false positive signals because of nonspecific binding of unrelated antibodies from human serum. LPS-free E. coli with autodisplayed La/SSB protein yielded sensitivity and selectivity of 81.6% and 78.6%, respectively. The Bland-Altman test showed that the immunoassays based on LPS-free E. coli and intact E. coli with autodisplayed La/SSB protein were statistically equivalent to a clinical immunoassay for detection of anti-La/SSB antibodies (confidence coefficient 95%).

Photoacoustic imaging probe for detecting lymph nodes and spreading of cancer at various depths.

We propose a compact and easy to use photoacoustic imaging (PAI) probe structure using a single strand of optical fiber and a beam combiner doubly reflecting acoustic waves for convenient detection of lymph nodes and cancers. Conventional PAI probes have difficulty detecting lymph nodes just beneath the skin or simultaneously investigating lymph nodes located in shallow as well as deep regions from skin without any supplementary material because the light and acoustic beams are intersecting obliquely in the probe. To overcome the limitations and improve their convenience, we propose a probe structure in which the illuminated light beam axis coincides with the axis of the ultrasound. The developed PAI probe was able to simultaneously achieve a wide range of images positioned from shallow to deep regions without the use of any supplementary material. Moreover, the proposed probe had low transmission losses for the light and acoustic beams. Therefore, the proposed PAI probe will be useful to easily detect lymph nodes and cancers in real clinical fields.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

This work presented a highly sensitive bacterial antibiotic susceptibility test through ß-lactamase assay using Parylene-matrix chip. ß-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to ß-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate ß-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The ß-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a ß-lactamase assay with minimal interference of low molecular weight noise peaks. The ß-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay.

Pore-size reduction protocol for SiN membrane nanopore using the thermal reflow in nanoimprinting for nanobio-based sensing.

Micro- and nano-fabrication methods facilitate the use of nanostructures for the separation of collections of particles and nanobio-based optical and electrochemical sensing. We have presented an easy and simple nanopore size reduction method of a low-stressed silicon nitride (SiN) membrane nanosieve (100×100 µm2) using a nanoimprinting method based on a natural thermal reflow of the contact imprinting polymer, possibly maintaining compatibility with complementary metal-oxide semiconductor integrated circuit processes. The nanopore pattern size of this nanosieve membrane was precisely patterned by a nanoimprinting process using an electron beam patterned silicon master, to about 30-nm diameter. By employing mainly an electron beam resist reflow phenomena after a nanoimprinting process and anisotropic reactive ion etch, the etch holes' size was fabricated to be the same with nanopatterns on the polymer. The contact imprinting master can be used continually for the generation of nanopore patterns simply and easily. It can endure harsh conditions like high temperature up to 800°C, and it is inert to many aggressive and strong chemicals. Also, this would be a low-cost, simple, and easy fabrication method for the precise and reliable size-reduction control of nanopores for mass production of nanobio sensors or chips.