Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis.

The global supply of fluoropolymers and fluorinated solvents is decreasing due to environmental concerns regarding polyfluoroalkyl substances. CYTOP has been used for decades primarily as a component of a femtoliter chamber array for digital bioanalysis; however, its supply has recently become scarce, increasing the urgency of fabricating a femtoliter chamber array using alternative materials. In this study, we investigated the feasibility of fabricating a femtoliter chamber array using four types of fluoropolymers in stable supply as candidate substitutes and verified their applicability for digital bioanalysis. Among these candidates, Fluorine Sealant emerged as a viable option for fabricating femtoliter chamber arrays using a conventional photolithography process. To validate its efficacy, we performed various digital bioanalysis using FP-A-based chamber arrays with model enzymes such as CRISPR-Cas, horseradish peroxidase, and ß-galactosidase. The results demonstrated the similar performance to that of CYTOP, highlighting the broader utility of FP-A in digital bioanalysis. Our findings underscore the potential of FP-A to enhance the versatility of digital bioanalysis and foster the ongoing advancement of innovative diagnostic technologies.

Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.

Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition.

Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single-molecule level, and it is shown that the dissecting alterations of single-molecule esterase activities can serve as an informative platform for activity-based diagnosis.

Toward versatile digital bioanalysis.

Digital bioanalysis places great emphasis on the highly sensitive and rapid detection of biomolecules at the single-molecule level. Rooted in single-molecule biophysics, this innovative approach offers numerous insights into biomolecular mechanisms with an unprecedented level of sensitivity and precision. Moreover, this method has significant potential to contribute to disease diagnostics, enabling the highly sensitive detection of biomarkers or pathogens for early disease diagnosis and continuous disease monitoring. However, the notable cost of detection and specialized equipment required for fabricating microdevices pose a challenge to accessibility and ease of use. This lack of versatility hinders the widespread adoption of digital bioanalysis. Here, we aim to illuminate the essential requirements for versatile digital bioanalysis and present prospects for biomedical applications that can be facilitated by attaining such versatility.

SATORI: Amplification-free digital RNA detection method for the diagnosis of viral infections.

With the recent global outbreak of COVID-19, there is an urgent need to establish a versatile diagnostic method for viral infections. Gene amplification test or antigen test are widely used to diagnose viral infections; however, these methods generally have technical drawbacks either in terms of sensitivity, accuracy, or throughput. To address this issue, we recently developed an amplification-free digital RNA detection method (SATORI), which can identify and detect viral genes at the single-molecule level in approximately 9 min, satisfying almost all detection performance requirements for the diagnosis of viral infections. In addition, we also developed practical platforms for SATORI, such as an automated platform (opn-SATORI) and a low-cost compact fluorescence imaging system (COWFISH), with the aim of application in clinical settings. Our latest technologies can be inherently applied to diagnose a variety of RNA viral infections, such as COVID-19 and Influenza A/B, and therefore, we expect that SATORI will be established as a versatile platform for point-of-care testing of a wide range of infectious diseases, thus contributing to the prevention of future epidemics. This article is an extended version of the Japanese article published in the SEIBUTSU BUTSURI Vol. 63, p. 115-118 (2023).

Purification/Amplification-Free RNA Detection Platform for Rapid and Multiplex Diagnosis of Plant Viral Infections.

Genetic tests are highly sensitive, and quantitative methods for diagnosing human viral infections, including COVID-19, are also being used to diagnose plant diseases in various agricultural settings. Conventional genetic tests for plant viruses are mostly based on methods that require purification and amplification of viral genomes from plant samples, which generally take several hours in total, making it difficult to use them in rapid detection at point-of-care testing (POCT). In this study, we developed Direct-SATORI, a rapid and robust genetic test that eliminates the purification and amplification processes of viral genomes by extending the recently developed amplification-free digital RNA detection platform called SATORI, allowing the detection of various plant viral genes in a total of less than 15 min with a limit of detection (LoD) of 98 ∼ copies/µL using tomato viruses as an example. In addition, the platform can simultaneously detect eight plant viruses directly from ∼1 mg of tomato leaves with a sensitivity of 96% and a specificity of 99%. Direct-SATORI can be applied to various infections related to RNA viruses, and its practical use is highly anticipated as a versatile platform for plant disease diagnostics in the future.

Development of fluorogenic substrates for colorectal tumor-related neuropeptidases for activity-based diagnosis.

The M3 metalloproteases, neurolysin and THOP1, are neuropeptidases that are expressed in various tissues and metabolize neuropeptides, such as neurotensin. The biological roles of these enzymes are not well characterized, partially because the chemical tools to analyse their activities are not well developed. Here, we developed a fluorogenic substrate probe for neurolysin and thimet oligopeptidase 1 (THOP1), which enabled the analysis of enzymatic activity changes in tissue and plasma samples. In particular, the probe was useful for studying enzyme activities in a single-molecule enzyme assay platform, which can detect enzyme activity with high sensitivity. We detected the activity of neurolysin in plasma samples and revealed higher enzyme activity in the blood samples of patients with colorectal tumor. The result indicated that single-molecule neurolysin activity is a promising candidate for a blood biomarker for colorectal cancer diagnosis.

Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance.

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.

Compact wide-field femtoliter-chamber imaging system for high-speed and accurate digital bioanalysis.

The femtoliter-chamber array is a bioanalytical platform that enables highly sensitive and quantitative analysis of biological reactions at the single-molecule level. This feature has been considered a key technology for "digital bioanalysis" in the biomedical field; however, its versatility is limited by the need for a large and expensive setup such as a fluorescence microscope, which requires a long time to acquire the entire image of a femtoliter-chamber array. To address these issues, we developed a compact and inexpensive wide-field imaging system (COWFISH) that can acquire fluorescence images with a large field of view (11.8 mm × 7.9 mm) and a high spatial resolution of ∼ 3 µm, enabling high-speed analysis of sub-million femtoliter chambers in 20 s. Using COWFISH, we demonstrated a CRISPR-Cas13a-based digital detection of viral RNA of SARS-CoV-2 with an equivalent detection sensitivity (limit of detection: 480 aM) and a 10-fold reduction in total imaging time, as compared to confocal fluorescence microscopy. In addition, we demonstrated the feasibility of COWFISH to discriminate between SARS-CoV-2-positive and -negative clinical specimens with 95% accuracy, showing its application in COVID-19 diagnosis. Therefore, COWFISH can serve as a compact and inexpensive imaging system for high-speed and accurate digital bioanalysis, paving a way for various biomedical applications, such as diagnosis of viral infections.

Hypercalcemia Associated with the Ectopic Expression of 25-hydroxyvitamin D3-1α-hydroxylase in Diffuse Large B-cell Lymphoma.

An 82-year-old man was transferred to our hospital due to impaired consciousness. His albumin-corrected calcium level was 14.2 mg/dL, intact parathyroid hormone (PTH) and PTH-related protein levels were reduced, and his 1,25-dihydroxyvitamin D [1,25 (OH) 2VitD] level was elevated at 71.5 pg/mL. Computed tomography revealed masses on the bilateral ribs. The mass on the rib was biopsied and diagnosed as diffuse large B-cell lymphoma (DLBCL). Immunostaining of the biopsy sample with the anti-CYP27B1 antibody revealed the ectopic expression of 1α-hydroxylase in the lesion. We herein report a rare case of hypercalcemia induced by the overproduction of 1,25 (OH) 2VitD in DLBCL ectopically expressing 1α-hydroxylase.

Automated amplification-free digital RNA detection platform for rapid and sensitive SARS-CoV-2 diagnosis.

In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of < 6.5 aM (3.9 copies µL-1), comparable to RT-qPCR. Additionally, opn-SATORI discriminated between SARS-CoV-2 variants of concern, including alpha, delta, and omicron, with 98% accuracy. Thus, opn-SATORI can serve as a rapid and convenient diagnostic platform for identifying several types of viral infections.

The association of high Vancomycin trough concentration with acute kidney injury during combination therapy of Piperacillin/Tazobactam and Vancomycin.

BACKGROUND: Co-administration of Piperacillin/Tazobactam (PIPC/TAZ) and Vancomycin (VCM) as an antibiotic therapy for severe infectious diseases increases the risk of nephrotoxicity. We retrospectively investigated the utility of monitoring VCM trough concentration in early stage of developing acute kidney injury (AKI) on this combination therapy. METHODS: We enrolled all infectious disease patients who were managed with concurrent PIPC/TAZ and VCM. The record of dosage and the administration interval of each antibiotic and its clinical parameters, as well as the VCM trough concentrations, blood culture for bacteria, and serum creatinine values, were collected. VCM trough concentration was measured during the initial 48-72 h of VCM administration. Nephrotoxicity was evaluated as the degree of AKI. RESULTS: A total of 47 patients fulfilling the criteria were registered, and AKI developed in 10 patients. There was no statistical difference between the AKI and non-AKI groups with regard to age, height, weight, basal creatinine level, body surface area, body mass index, PIPC/TAZ dose, VCM dose, gender, artificial management, and death within around 30 days. The VCM trough level was increased significantly in the AKI group (mean [standard deviation {SD}]: 25.9 [7.8] µg/mL) compared to that in the non-AKI group (mean [SD]: 15.7 [6.9] µg/mL) (p = 0.003). During the clinical course, renal function returned to normal levels in three out of four AKI stage 2 patients, whereas only partial recovery was achieved in all AKI stage 3 patients. CONCLUSIONS: A high VCM trough concentration may have an influence on the occurrence of AKI during combination therapy of PIPC/TAZ and VCM. Careful monitoring of VCM trough concentration will be required to prevent AKI progression.

Amplification-free RNA detection with CRISPR-Cas13.

CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.

Monodisperse Liposomes with Femtoliter Volume Enable Quantitative Digital Bioassays of Membrane Transporters and Cell-Free Gene Expression.

Digital bioassays have emerged as a new category of bioanalysis. However, digital bioassays for membrane transporter proteins have not been well established yet despite high demands in molecular physiology and molecular pharmacology due to the lack of biologically functional monodisperse liposomes with femtoliter volumes. Here, we established a simple and robust method to produce femtoliter-sized liposomes (femto-liposomes). We prepared 106 monodispersed water-in-oil droplets stabilized by a lipid monolayer using a polyethylene glycol-coated femtoliter reactor array device. Droplets were subjected to the optimized emulsion transfer process for femto-liposome production. Liposomes were monodispersed (coefficient of variation = 5-15%) and had suitable diameter (0.6-5.3 µm) and uniform volumes of subfemtoliter or a few femtoliters; thus, they were termed uniform femto-liposomes. The unilamellarity of uniform femto-liposomes allowed quantitative single-molecule analysis of passive and active transporter proteins: α-hemolysin and FoF1-ATPase. Digital gene expression in uniform femto-liposomes (cell-free transcription and translation from single DNA molecules) was also demonstrated, showing the versatility of digital assays for membrane transporter proteins and cell-free synthetic biology.

Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples.

We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we "count" the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity-based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester-hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.

Microsystem for the single molecule analysis of membrane transport proteins.

Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level. Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although various functions of membrane proteins are extremely important. To address this issue, microsystems using arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a "biomembrane microsystems", have been developed by many research groups for the analysis of membrane proteins. In this review, I would like to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomembrane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.

High-throughput single-molecule bioassay using micro-reactor arrays with a concentration gradient of target molecules.

Micro-reactor arrays enable highly sensitive and quantitative bioassays at a single-molecule level. Accordingly, they are widely used for sensitive "digital" bioassays, e.g., digital PCR and digital ELISA. Despite high integration, individual reactors in digital bioassays are filled with a uniform reaction solution, thus limiting the ability to simultaneously conduct multiple bioassays under different conditions using integrated reactors in parallel, resulting in the loss of potential throughput. We developed micro-reactor arrays with a concentration gradient of target molecules, in which individual reactors sealed with a lipid-bilayer membrane contained a precise amount of target molecules. Using the arrays, we successfully demonstrated multiple single-molecule bioassays in parallel using alkaline phosphatase or α-hemolysin, key components in various biomedical sensors. This new platform extends the versatility of micro-reactor arrays and could enable further analytical and pharmacological applications.

Hybrid cell reactor system from Escherichia coli protoplast cells and arrayed lipid bilayer chamber device.

We developed a novel hybrid cell reactor system via functional fusion of single Escherichia coli protoplast cells, that are deficient in cell wall and expose plasma membrane, with arrayed lipid bilayer chambers on a device in order to incorporate the full set of cytosolic and membrane constituents into the artificial chambers. We investigated gene expression activity to represent the viability of the hybrid cell reactors: over 20% of hybrid cells showed gene expression activity from plasmid or mRNA. This suggests that the hybrid cell reactors retained fundamental activity of genetic information transduction. To expand the applicability of the hybrid cell reactors, we also developed the E. coli-in-E. coli cytoplasm system as an artificial parasitism system. Over 30% of encapsulated E. coli cells exhibited normal cell division, showing that hybrid cells can accommodate and cultivate living cells. This novel artificial cell reactor technology would enable unique approaches for synthetic cell researches such as reconstruction of living cell, artificial parasitism/symbiosis system, or physical simulation to test functionality of synthetic genome.

Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance.

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1-elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin-adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

Single-molecule analysis of phospholipid scrambling by TMEM16F.

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.