Streamlined Specimen Purification for Rapid COVID-19 Diagnosis Using Positively Charged Polymer Thin Film-Coated Surfaces and Chamber Digital PCR.

The ongoing coronavirus disease 2019 (COVID-19) pandemic demands rapid and straightforward diagnostic tools to prevent early-stage viral transmission. Although nasopharyngeal swabs are a widely used patient sample collection method for diagnosing COVID-19, using these samples for diagnosis without RNA extraction increases the risk of obtaining false-positive and -negative results. Thus, multiple purification steps are necessary, which are time-consuming, generate significant waste, and result in substantial sample loss. To address these issues, we developed surface-modified polymerase chain reaction (PCR) tubes using the tertiary aminated polymer poly(2-dimethylaminomethylstyrene) (pDMAMS) via initiated chemical vapor deposition. Introducing the clinical samples into the pDMAMS-coated tubes resulted in approximately 100% RNA capture efficiency within 25 min, which occurred through electrostatic interactions between the positively charged pDMAMS surface and the negatively charged RNA. The captured RNA is then detected via chamber digital PCR, enabling a sensitive, accurate, and rapid diagnosis. Our platform provides a simple and efficient RNA extraction and detection strategy that allows detection from 22 nasopharyngeal swabs and 21 saliva specimens with 0% false negatives. The proposed method can facilitate the diagnosis of COVID-19 and contribute to the prevention of early-stage transmission.

Sensitive and non-invasive cholesterol determination in saliva via optimization of enzyme loading and platinum nano-cluster composition.

An excessive cholesterol level can lead to cardiovascular diseases, such as stroke, hypertension, and myocardial infarction. A non-invasive, painless method of determining the cholesterol level in blood would improve the user's convenience. To provide rapid and accurate determination of cholesterol, we have developed a simple, disposable, enzyme-based electrochemical biosensor that can detect salivary cholesterol. It is possible to detect low concentrations of cholesterol in saliva using the optimized vertical structure of the platinum nano-cluster (Pt-NC) and the immobilization of a proper volume of an enzyme. The biosensor exhibited a linear range from 2 to 486 µM, the limit of detection was about 2 µM, and the sensitivity of the sensor was calculated to be 132 µA mM-1 cm-2. It also showed good specificity for ascorbic acid, uric acid, dopamine, glucose, and lactate. In a test with an actual sample, the performance of the biosensor was confirmed by measuring total cholesterol in the saliva of a patient with hyperlipidemia. The cholesterol levels measured in the saliva of three patients with hyperlipidemia were 520, 460, and 290 µM. Therefore, the Pt-NC based enzyme sensor is a promising candidate for the detection of cholesterol in human saliva.

Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva.

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Tunable and scalable fabrication of block copolymer-based 3D polymorphic artificial cell membrane array.

Owing to their excellent durability, tunable physical properties, and biofunctionality, block copolymer-based membranes provide a platform for various biotechnological applications. However, conventional approaches for fabricating block copolymer membranes produce only planar or suspended polymersome structures, which limits their utilization. This study is the first to demonstrate that an electric-field-assisted self-assembly technique can allow controllable and scalable fabrication of 3-dimensional block copolymer artificial cell membranes (3DBCPMs) immobilized on predefined locations. Topographically and chemically structured microwell array templates facilitate uniform patterning of block copolymers and serve as reactors for the effective growth of 3DBCPMs. Modulating the concentration of the block copolymer and the amplitude/frequency of the electric field generates 3DBCPMs with diverse shapes, controlled sizes, and high stability (100% survival over 50 days). In vitro protein-membrane assays and mimicking of human intestinal organs highlight the potential of 3DBCPMs for a variety of biological applications such as artificial cells, cell-mimetic biosensors, and bioreactors.

Rapid exchange of oil-phase in microencapsulation chip to enhance cell viability.

This paper describes a microfluidic device for the microencapsulation of cells in alginate beads to enhance cell viability. The alginate droplet including cells was gelified with calcified oleic acid, using two-phase microfluidics. The on-chip gelation had generated monodisperse spherical alginate beads, which could not be readily obtained via conventional external gelation in a calcium chloride bath. However, the prolonged exposure of encapsulated cells to the toxic oil phase caused serious damage to the cells. Therefore, we proposed the formulation of a rapid oil-exchange chip which transforms the toxic oleic acid to harmless mineral oil. The flushing out of oleic acid after the gelation of alginate beads effected a dramatic increase in the viability of P19 embryonic carcinoma cells, up to 90%. The experimental results demonstrated that the cell viability was proportional to the flow rate of squeezing mineral oil.

Oil droplet generation in PDMS microchannel using an amphiphilic continuous phase.

This paper reports an amphiphilic solution can be used as a new continuous phase to generate double droplet emulsions (water/oil/IPA) with neither surface treatment nor surfactant in PDMS microfluidic chip. The affinity of various amphiphilic solutions in the microchannel was influenced by the polarity ratio and the size of molecules. The polarity ratio of isopropyl alcohol (IPA) was closest to that of the recovered PDMS surface and the chain length of IPA was also suitable for high affinity. IPA showed the highest affinity for the recovered PDMS and was selected as the continuous phase to form oil droplets in a PDMS microchannel. With this new continuous phase solution, IPA, we could successfully generate not only oil droplets but also double emulsions in the PDMS microfluidic chips.

A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics.

An innovative polymer lab-on-a-chip (LOC) for reverse transcription (RT)-polymerase chain reaction (PCR) has been designed, fabricated, and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer that consists of a non-contact infrared (IR) based temperature control system for RT-PCR process and an optical detection system for on-chip detection, has also been developed and used to monitor the RT-PCR LOC. The newly developed LOC and analyzer have been interfaced and optimized for performing RT-PCR procedures and chemiluminescence assays in sequence. As a clinical diagnostic application, human immunodeficiency virus (HIV) for the early diagnosis of acquired immune deficiency syndrome (AIDS) has been successfully detected and analyzed using the newly developed LOC and analyzer, where the primer sets for p24 and gp120 were used as the makers for HIV. The developed polymer LOC and analyzer for RT-PCR can be used for POCT for the analysis of HIV with the on-chip RT-PCR and chemiluminescence assays in shorter than one hour with minimized cross-contamination.

Hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease.

Quantitative polymerase chain reaction (qPCR) renders profiling of genes of interest less time-consuming and cost-effective. Recently, multiplex profiling of miRNAs has enabled identifying or investigating predominant miRNAs for various diseases such as cancers and neurodegenerative diseases. Conventional multiplex qPCR technologies mostly use colorimetric measurements in solution phase, yet not only suffer from limited multiplexing capacity but also require target-screening processes due to non-specific binding between targets and primers. Here, we present hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease (AD). Our methodology promises two key advantages compared with the conventional solution-based PCR: 1) nearly no non-specific crosstalks between targets and primers, and 2) practically valuable multiplexing by spatial encoding within a single microchamber. Specifically, we immobilized hydrogel microposts (~ 400µm in diameter) within commercially available polycarbonate PCR chips by multi-step ultraviolet (UV, 365nm) exposure. We optimized this photoimmobilization for thermal cycles of PCR as well. Acrylated forward primers incorporated in polyethylene glycol diacrylate (PEGDA) posts played a crucial role to confine fluorescent signal of cDNA amplification within the PEGDA hydrogel. To demonstrate the potential of our platform, we successfully verified multiplex detection of five miRNAs, which were reported to be highly correlated with AD, from a complex buffer of human plasma.

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

Biofunctionalization of Nerve Interface via Biocompatible Polymer-Roughened Pt Black on Cuff Electrode for Chronic Recording.

Peripheral nerve cuff electrodes with roughened Pt black (BPt) are coated with polyethylene glycol (PEG) and Nafion (NF). Although the influence of coated PEG and Nafion on roughened BPt on the electrical properties is weak, the cuff electrode with BPt/PEG and BPt/Nafion exhibits some very important properties. For example, it markedly decreases interfacial impedance, increases charge storage capacity (CSC) due to retaining the BPt surface structure, good stability without exfoliation in repetitive cyclic voltammetry scanning because it is protected by PEG or Nafion coating. In cell viability test, Nafion-coated BPt does not show cytotoxicity to rat Schwann cell line (S16) at 24 and 72 h with the Nafion coating ranging from 0.1 to 10 mg cm-2 . In addition, real-time polymerase chain reaction (PCR) analysis indicates that Schwann cell differentiation (S100 calcium-binding protein B, myelin basic protein, peripheral myelin protein 22), proliferation (proliferating cell nuclear antigen, cyclin-dependent kinase 1 (CDK1)), and adhesion molecules (neural cell adhesion molecule, laminin, fibronectin) are upregulated up to 5 mg cm-2 of Nafion. In animal study, the BPt/Nafion reduces infiltration of fibrotic tissue with high axonal maintenance with upregulation of proliferation (CDK1), adhesion (laminin, neuronal cell adhesion molecule), and neurotrophic factor receptor-related (gdnf family receptor alpha 1) mRNA expressions.

Biological assessments of multifunctional hydrogel-decorated implantable neural cuff electrode for clinical neurology application.

The implantable cuff electrode is an effective neuroprosthetic device in current nerve tissue engineering. However, biocompatibility and stability are still a serious dispute in terms of in vivo function and continuous monitoring. In this regard, assessing the host's biological response to biomaterials is one of the key factors of chronic implantation. In this article, we analyzed the peripheral nerve specific-biological responses to the application of multi-functional hydrogel-coated electrodes. The surface of the cuff electrode was modified using a multifunctional hydrogel composed of PEG hydrogel, cyclosporin A(CsA)-microsphere(MS) and electrodeposited PEDOT:PSS. Through our approach, we have found that the multifunctional hydrogel coatings improve the neural electrode function, such as peak-to-peak amplitude increase. Additionally, the multifunctional hydrogel coated electrodes exhibited improved biocompatibility, such as reduced apoptotic properties and increased axonal myelination. Furthermore, 12 genes (BDNF, Gfra1, IL-6, Sox 10, S100B, P75 NTR , GAP43, MBP, MPZ, NrCAM, NE-FL, CB1) were upregulated at 5 weeks post-implant. Finally, double immunofluorescence revealed the effect of endocannabinoid system on neuroprotective properties and tissue remodeling of peripheral nerves during cuff electrode implantation. These results clearly confirmed that multifunctional hydrogel coatings could improve electrode function and biocompatibility by enhancing neuroprotective properties, which may provide a valuable paradigm for clinical neurology application.

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (µCHAMPs) for the detection of amyloid beta oligomers.

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 µL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (µCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this µCHAMP-based immunoassay platform, small volumes (30-50 µL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 µm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this µCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aß) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 µL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aß oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the µCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

Multifunctional hydrogel coatings on the surface of neural cuff electrode for improving electrode-nerve tissue interfaces.

UNLABELLED: Recently, implantable neural electrodes have been developed for recording and stimulation of the nervous system. However, when the electrode is implanted onto the nerve trunk, the rigid polyimide has a risk of damaging the nerve and can also cause inflammation due to a mechanical mismatch between the stiff polyimide and the soft biological tissue. These processes can interrupt the transmission of nerve signaling. In this paper, we have developed a nerve electrode coated with PEG hydrogel that contains poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with anti-inflammatory cyclosporin A (CsA). Micro-wells were introduced onto the electrode in order to increase their surface area. This allows for loading a high-dose of the drug. Additionally, chemically treating the surface with aminopropylmethacrylamide can improve the adhesive interface between the electrode and the hydrogel. The surface of the micro-well cuff electrode (MCE) coated with polyethylene glycol (PEG) hydrogel and drug loaded PLGA microspheres (MS) were characterized by SEM and optical microscopy. Additionally, the conductive polymers, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT/PSS), were formed on the hydrogel layer for improving the nerve signal quality, and then characterized for their electrochemical properties. The loading efficiencies and release profiles were investigated by High Performance Liquid Chromatography (HPLC). The drug loaded electrode resulted in a sustained release of CsA. Moreover, the surface coated electrode with PEG hydrogel and CsA loaded MP showed a significantly decreased fibrous tissue deposition and increased axonal density in animal tests. We expect that the developed nerve electrode will minimize the tissue damage during regeneration of the nervous system. STATEMENT OF SIGNIFICANCE: The nerve electrodes are used for interfacing with the central nervous system (CNS) or with the peripheral nervous system (PNS). The interface electrodes should facilitate a closed interconnection with the nerve tissue and provide for selective stimulation and recording from multiple, independent, neurons of the neural system. In this case, an extraneural electrodes such as cuff and perineural electrodes are widely investigated because they can completely cover the nerve trunk and provide for a wide interface area. In this study, we have designed and prepared a functionalized nerve cuff electrode coated with PEG hydrogel containing Poly lactic-co-glycol acid (PLGA) microspheres (MS) loaded with cyclosporine A (CsA). To our knowledge, our findings suggest that surface coating a soft-hydrogel along with an anti-inflammatory drug loaded MS can be a useful strategy for improving the long-term biocompatibility of electrodes.

Fabrication and characterization of stimulus nerve cuff electrode with highly roughened surface for chronic implant.

Nerve cuff electrodes for peripheral nerve prostheses are required chronically implanted electrodes which simultaneously stimulate and record nerve activity. It is inevitable challenge to investigate electrode material with low interfacial impedance and enhanced charge transfer capacity. In this study, stimulus nerve cuff electrodes on polyimide with Pt, conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT), black Pt, and IrOx were fabricated and characterized. The electrochemical properties were investigated using cyclic voltammetry, electrochemical impedance spectroscopy, and voltage transient measurements. From th experimental results, stimulus nerve cuff electrodes with black Pt showed the highest charge delivery capacity (80 times higher than Pt), charge injection capacity (6 times higher than Pt), and lowest interfacial impedance (3.8 times lower than Pt).

Characterization of proteolytically digested zebrafish chorion as extracellular matrix.

Extracellular matrix (ECM) plays pivotal roles in developments, wound healing, migration, angiogenesis, and other tissue developments. Developing new ECM may give us an innovative approach of controlling the cell and tissue behavior, regenerating damaged tissues, and other biotechnology usages. Here we report new ECM like biomaterials, pFZC, obtained from the fertilized Zebrafish chorion (FZC). pFZC was prepared by digesting the fertilized Zebrafish chorion with pronase. Differentiation of P19 embryonic carcinoma cells (P19 EC) on pFZC demonstrated that the level of the differentiation of P19 EC was comparable to cells on gelatin or fibronectin coated cover slide. Analyses of glycosylations of Zebrafish chorions using various Lectins revealed that during the fertilization, structurally organized glycosylations of Zebrafish chorion were increased and FZC was glycosylated with mannose, fucose, n-acetylglucosamine, and sialic acid. In addition, after FZC proteolytic digestion with pronase, the digested FZC proteins, pFZC, displayed similar glycosylation manner. SDS gel electrophoresis and mass spectroscopy analysis of pFZC revealed that the major component of pFZC is ZP2 protein. Newly identified ECM like biomaterials, pFZC, will provide more broad ways of controlling the cell and tissue environments.

Using the chorions of fertilized zebrafish eggs as a biomaterial for the attachment and differentiation of mouse stem cells.

The development of proper biomaterials is critical for the success of cell therapy and modern tissue engineering. Here, we extruded the yolk and remaining inner mass from fertilized zebrafish eggs and used the resulting chorions as a biomaterial for the differentiation and attachment of mouse P19 embryonic carcinoma (EC) cells. Cells inserted into the chorion showed the spontaneous formation of embryoid body due to the repulsive cell adhesion of the chorion and differentiated specifically into neural cells and cardiomyocytes. In contrast, dissolved chorion extracellular matrix (ECM) conferred enhanced cell attachment on it, suggesting that a unique property of the zebrafish chorion with nanoporous structure appears to be responsible for the simple and controllable embryoid formation for stem cell differentiation. These results indicate that chorions from fertilized zebrafish eggs may be used as an extracellular matrix alternative and applied for stem cell differentiation to specific cell lineages.