Water detection in organic solvents using a copolymer membrane immobilised with a fluorescent intramolecular charge transfer-type dye: effects of intramolecular hydrogen bonds.

Numerous fluorescent dye-based optical sensors have been developed to detect water in organic solvents. However, only a few such sensors can detect water in polar solvents such as methanol or dimethyl sulfoxide, and their detection range is generally narrow. Therefore, in this study, a copolymer membrane incorporated with a pyridinium betaine dye (denoted PB1), which exhibited intramolecular charge transfer (ICT) characteristics, was developed to realise simple water detection in organic solvents. The pyridinium betaine structure, comprising intramolecular hydrogen bonds between the oxygen in the maleimide moiety and the hydrogen in the pyridinium, was vital for achieving efficient fluorescence emission. The membrane was prepared by copolymerising PB1 with the N,N-dimethyl acrylamide/acrylamide monomer on a glass plate, and the fluorescence in water-mixed organic solvents was investigated (λabs = 490 nm, λfl = 630 nm). The fluorescence intensity of the dye-immobilised membrane decreased with increasing water content of the organic solvents. The detection ranges in tetrahydrofuran, ethanol, methanol, and dimethyl sulfoxide were approximately <40, <40, <40, and <60 vol% water, respectively. In contrast, membranes based on a quaternary pyridinium dye (without intramolecular hydrogen bonds) did not detect water in methanol and dimethyl sulfoxide, although it was more sensitive than PB1 in the narrow region of low water concentration in THF. Theoretical calculations corroborated the importance of the pyridinium betaine structure in detecting water in organic solvents, with the increase in polarity and the formation of intermolecular hydrogen bonds between PB1 and water found to induce molecular rotation and fluorescence quenching.

Assessment of the structural change of DNA by binding with a small molecule based on capillary sieving electrophoresis.

In this study, capillary sieving electrophoresis (CSE) using polymer solutions was used to evaluate the structural changes in nucleic acids upon complexation with small molecules. As the model target and nucleic acids, L-tyrosinamide (Tyr-Am) and its aptamer, which is a type of DNA specifically binding to Tyr-Am, were selected. CSE was conducted using a capillary filled with background solution (BGS) containing hydroxypropyl cellulose (HPC) as a sieving matrix. When Tyr-Am or tyrosine was added to the BGS in CSE, the ratio of mobility differences of the Tyr-Am-aptamer complex increased compared to that of the free aptamer without the addition of Tyr-Am. In contrast, when other amino acids or their analogs were added, results showed no apparent change or decreases in electrophoretic mobility. These results indicate that the proposed method can be applied to assess structural changes in nucleic acids that target small molecules.

Dielectrophoretic trapping of nanosized biomolecules on plasmonic nanohole arrays for biosensor applications: simple fabrication and visible-region detection.

Surface plasmon resonance is an optical phenomenon that can be applied for label-free, real-time sensing to directly measure biomolecular interactions and detect biomarkers in solutions. Previous studies using plasmonic nanohole arrays have monitored and detected various biomolecules owing to the propagating surface plasmon polaritons (SPPs). Extraordinary optical transmission (EOT) that occurs in the near-infrared (NIR) and infrared (IR) regions is usually used for detection. Although these plasmonic nanohole arrays improve the sensitivity and throughput for biomolecular detection, these arrays have the following disadvantages: (1) molecular diffusion in the solution (making the detection of biomolecules difficult), (2) the device fabrication's complexities, and (3) expensive equipments for detection in the NIR or IR regions. Therefore, there is a need to fabricate plasmonic nanohole arrays as biomolecular detection platforms using a simple and highly reproducible procedure based on other SPP modes in the visible region instead of the EOT in the NIR or IR regions while suppressing molecular diffusion in the solution. In this paper, we propose the combination of a polymer-based gold nanohole array (Au NHA) obtained through an easy process as a simple platform and dielectrophoresis (DEP) as a biomolecule manipulation method. This approach was experimentally demonstrated using SPP and LSPR modes (not EOT) in the visible region and simple, label-free, rapid, cost-effective trapping and enrichment of nanoparticles (trapping time: <50 s) and bovine serum albumin (trapping time: <1000 s) was realized. These results prove that the Au NHA-based DEP devices have great potential for real-time digital and Raman bioimaging, in addition to biomarker detection.

Nanoemulsion-based silver ion-selective optode based on colorimetrically silver ion-responsive ionic liquid-based dye.

In this study, we describe the fast-responsive nanoemulsion (NE)-based silver ion (Ag+)-selective optode based on colorimetrically silver ion-responsive ionic liquid-based dye (ILD). The ILD comprises purely functional sensing molecules, a protonated cationic merocyanine dye (KD-M13-H+) and an anionic Ag+ ionophore (BDM-SO3-), and thus, it can be used for highly sensitive silver ion (Ag+) sensing due to the extremely high content of dye in the organic phase (ionic-liquid phase). However, during the Ag+ sensing, the cationic merocyanine dye is converted into electrically neutral form by deprotonation of the dye, which leads to the conversion of liquified dye into solid form in the organic phase, which makes the response time slower when ILD is used for poly(vinyl chloride) (PVC) membrane-based ion-selective optode, especially for sensing of high Ag+ concentration. To solve this problem, we focused on the use of the nano-emulsification technique. The response time of the ILD-based nanoemulsion (NE) was considerably shorter (1 s) compared to that of the ILD-based PVC membrane (a few minutes) owing to the large surface area and excellent diffusivity of the emulsion. The ILD-based NE contained a very high dye concentration (833 mmol kg-1) and exhibited approximately 12 times higher sensitivity than that of the plasticizer-based conventional NE. In the cation measurements, the ILD-based NE responded to Ag+ via a cation-exchange mechanism and demonstrated a highly selective response to Ag+ (log [Formula: see text] = - 3.0). ILD-NE was successfully applied to the detection of spiked Ag+ in a tap water sample with recoveries of 98 - 103% with a relative standard deviation (RSD) of less than 5%. In comparison with NE based on non-ionic ionophores without charge, NE based on BDM-SO3- responded to lower Ag+ concentrations owing to the effect of negative charge on the binding property. The novel ILD-based NE was capable of highly sensitive, rapid, and selective Ag+ sensing, providing potential for analytical devices applicable to high-performance on-site analysis.

Fabrication of Gold Nanostructures on Quartz Crystal Microbalance Surface Using Nanoimprint Lithography for Sensing Applications.

A quartz crystal microbalance (QCM) is a sensor that uses the piezoelectric properties of quartz crystals sandwiched between conductive electrodes. Localized surface plasmon resonance (LSPR) is an analytical technique that uses the collective vibration of free electrons on metal surfaces. These measurements are known as analysis techniques that use metal surfaces and have been applied as biosensors because they allow for the label-free monitoring of biomolecular binding reactions. These measurements can be used in combination to analyze the reactions that occur on metal surfaces because different types of information can be obtained from them. However, as different devices are used for these measurements, the results often contain device-to-device errors and are not accurately evaluated. In this study, we directly fabricated gold nanostructures on the surface of a QCM to create a device that can simultaneously measure the mass and refractive index information of the analyte. In addition, the device could be easily fabricated because nanoimprint lithography was used to fabricate gold nanostructures. As a proof of concept, the nanoparticle adsorption on gold nanostructures was evaluated, and it was observed that mass and refractive index information were successfully obtained without device-to-device errors.

Aptamer Selection Based on Microscale Electrophoretic Filtration Using a Hydrogel-Plugged Capillary Device.

This study reports a novel aptamer selection method based on microscale electrophoretic filtration. Aptamers are versatile materials that recognize specific targets and are attractive for their applications in biosensors, diagnosis, and therapy. However, their practical applications remain scarce due to issues with conventional selection methods, such as complicated operations, low-efficiency separation, and expensive apparatus. To overcome these drawbacks, a selection method based on microscale electrophoretic filtration using a capillary partially filled with hydrogel was developed. The electrophoretic filtration of model target proteins (immunoglobulin E (IgE)) using hydrogel, the electrokinetic injection of DNAs to interact with the trapped proteins, the elimination of DNAs with weak interactions, and the selective acquisition of aptamer candidates with strong interactions were successfully demonstrated, revealing the validity of the proposed concept. Two aptamer candidates for IgE were obtained after three selection cycles, and their affinity for the target was confirmed to be less than 1 nM based on their dissociation constant (KD) values. Therefore, the proposed method allows for the selection of aptamers with simple operations, highly effective separation based on electrophoresis and filtration, and a relatively cheap apparatus with disposable devices.

Gold Nanocone Array with Extensive Electromagnetic Fields for Highly Reproducible Surface-Enhanced Raman Scattering Measurements.

Surface-enhanced Raman scattering (SERS) is a technique used to distinguish the constitution of disease-related biomarkers in liquid biopsies, such as exosomes and circulating tumor cells, without any recognition elements. Previous studies using metal nanoparticle aggregates and angular nanostructures have achieved the detection of various biomarkers owing to strong hot spots and electromagnetic (EM) fields by localized surface plasmon resonance (LSPR). Although these SERS platforms enable significant enhancement of Raman signals, they still have some problems with the fabrication reproducibility of platforms in obtaining reproducible SERS signals. Therefore, highly reproducible fabrication of SERS platforms is required. Here, we propose the application of a polymer-based gold (Au) nanocone array (Au NCA), which extensively generates an enhanced EM field near the Au NCA surface by LSPR. This approach was experimentally demonstrated using a 785 nm laser, typically used for SERS measurements, and showed excellent substrate-to-substrate reproducibility (relative standard deviation (RSD) < 6%) using an extremely simple fabrication procedure and very low laser energy. These results proved that a Au NCA can be used as a highly reproducible SERS measurement to distinguish the constitution of biomarkers.

Development of transient trapping micellar electrokinetic chromatography coupled with mass spectrometry for steroids analysis.

An on-line sample preconcentration technique based on transient trapping (tr-trapping) in micellar electrokinetic chromatography (MEKC) was applied for steroid detection with UV (tr-trapping-UV) and electrospray ionization mass spectrometry detection (tr-trapping-ESI-MS). ESI-MS was used to improve the sensitivity in MEKC. The MEKC separation was carried out using volatile ammonium formate as a background solution to facilitate the coupling with ESI-MS. The partial introduction of a sodium dodecyl sulfate (SDS) micellar solution before the introduction of a sample solution to the capillary provided the effective preconcentration of analytes. At the same time, the SDS micelle would not enter the ESI-MS system, so its interference in ESI-MS detection was suppressed under the optimal condition, then five steroids can be separated by the developed method. In tr-trapping-ESI-MS, an acidic condition of pH 3.5 was employed to suppress the electroosmotic flow, which can avoid micellar solution migrating to the MS instrument. The developed method showed that the micellar solution requires a twofold slower time than the sample to migrate along the column, which can prohibit the cause of the problem with the MS instrument and interference signal of SDS in the steroid's detection. The tr-trapping-ESI-MS protocol showed up to 540-fold enhancements of the peak intensity and 50-fold improvement of the limit of detection compared with capillary zone electrophoresis using androsterone as a model sample.

Imprinted Photonic Crystal-Film-Based Smartphone-Compatible Label-Free Optical Sensor for SARS-CoV-2 Testing.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 has caused a global pandemic. To manage and control the spread of the infection, it is crucial to develop and implement technologies for the early identification of infected individuals and rapid informatization in communities. For the realization of such a technology, a widely available and highly usable sensor for sensitive and specific assay of the virus plays a fundamental role. In this study, we developed an optical sensor based on an imprinted photonic crystal film (IPCF) for quick, simple, and cost-effective detection of SARS-CoV-2 spike protein in artificial saliva. Our IPCF sensor enabled label-free and highly sensitive detection with a smartphone-equipped optical setup. The IPCF surface was functionalized with an anti-SARS-CoV-2 spike protein antibody for immunoassay. We evaluated the specificity and sensitivity of the IPCF sensor for quantitative detection of the spike protein in artificial saliva using simple reflectometry with a spectrometer-equipped optical setup. Specific and quantitative detection of the spike protein was successfully achieved, with a low detection limit of 429 fg/mL. In the demonstration of reflectometric detection with a smartphone-equipped setup, the sensitivity was comparable with that with a spectrometer-equipped setup. The test result is returned immediately and can be saved to cloud storage. In addition, it costs less than USD 1 for one IPCF to be used for diagnosis. Thus, the developed IPCF has the potential to realize a widely available and highly usable sensor.

Evaluation of the interactions between oligonucleotides and small molecules by partial filling-nonequilibrium affinity capillary electrophoresis.

Recently, high-throughput analysis with minimal reagent consumption has been desired to assess interactions between drug candidates and disease-related oligonucleotides. To realize an ideal assay for drug screening, a rapid assay based on affinity capillary electrophoresis was generated to reduce the consumption of samples/reagents by a partial-filling technique under nonequilibrium conditions. In the proposed method, the first sample, oligonucleotide as a ligand, and second sample zones were injected into a capillary with spacers of background solution between the samples and oligonucleotide zones. After applying voltages, only the second sample zone passed through the partially filled oligonucleotide zone, resulting in variations in the peak parameters owing to this interaction. The electropherograms obtained were analyzed using equilibrium, reaction kinetics, and moment theories. In the interaction analyses between small molecules and DNA aptamers, only the small molecules binding to the aptamer showed significant changes in their peak heights and shapes. The estimated kinetic parameters were in good agreement with the reported values, indicating the applicability of the proposed method for drug screening. When interactions between drug candidates and disease-related RNAs were analyzed, one of the candidates showed remarkable variation in the peak parameters upon the addition of potassium ions. Consequently, the proposed method could be one of the ideal assays for drug screening.

Chloride ion-selective dye liquid nanoemulsion: improved sensor performance due to intermolecular interactions between dye and ionophore.

Ionophore-based dye liquid nanoemulsion sensors exhibiting rapid response, high selectivity, and high sensitivity to chloride were developed. Since nanoemulsions contain extremely high concentrations of dyes and have large surface areas, rapid and highly sensitive measurements were possible. 1H-NMR measurements revealed intermolecular interactions between the dye and the ionophore, which contributed to the suppression of the background signal.

Fabrication of Metal-Insulator-Metal Nanostructures Composed of Au-MgF2-Au and Its Potential in Responding to Two Different Factors in Sample Solutions Using Individual Plasmon Modes.

In this paper, metal-insulator-metal (MIM) nanostructures, which were designed to exhibit two absorption peaks within 500-1100 nm wavelength range, were fabricated using magnesium difluoride (MgF2) as the insulator layer. Since the MIM nanostructures have two plasmon modes corresponding to the absorption peaks, they independently responded to the changes in two phases: the surrounding medium and the inside insulator layer, the structure is expected to obtain multiple information from sample solution: refractive index (RI) and molecular interaction between solution components and the insulator layer. The fabricated MIM nanostructure had a diameter of 139.6 ± 2.8 nm and a slope of 70°, and exhibited absorption peaks derived from individual plasmon modes at the 719 and 907 nm wavelengths. The evaluation of the response to surrounding solution component of the MIM nanostructures revealed a linear response of one plasmon mode toward the RI of the surrounding medium and a large blue shift of the other plasmon mode under conditions where glycerol was present at high concentration. From optical simulation and the evaluation of the MgF2 fabricated by deposition, the blue shift was expected to be due to the swelling of MgF2 interacting with the hydroxyl groups abundantly included in the glycerol molecules. The results indicated the individual responses of two plasmon modes in MIM nanostructures toward medium components, and brought the prospect for the simultaneous measurement of multiple elements using two or more plasmon modes.

Modulating Optical Characteristics of Nanoimprinted Plasmonic Device by Re-Shaping Process of Polymer Mold.

Metal nanostructures exhibit specific optical characteristics owing to their localized surface plasmon resonance (LSPR) and have been studied for applications in various optical devices. The LSPR property strongly depends on the size and shape of metal nanostructures; thus, plasmonic devices must be designed and fabricated according to their uses. Nanoimprint lithography (NIL) is an effective process for repeatedly fabricating metal nanostructures with controlled sizes and shapes and require optical properties. NIL is a powerful method for mass-producible, low-cost, and large-area fabrication. However, the process lacks flexibility in adjusting the size and shape according to the desirable optical characteristics because the size and shape of metal nanostructures are determined by a single corresponding mold. Here, we conducted a re-shaping process through the air-plasma etching of a polymer's secondary mold (two-dimensional nanopillar array made of cyclo-olefin polymer (COP)) to modulate the sizes and shapes of nanopillars; then, we controlled the spectral characteristics of the imprinted plasmonic devices. The relationship between the structural change of the mold, which was based on etching time, and the optical characteristics of the corresponding plasmonic device was evaluated through experiments and simulations. According to evaluation results, the diameter of the nanopillar was controlled from 248 to 139 nm due to the etching time and formation of a pit structure. Consequently, the spectral properties changed, and responsivity to the surrounding dielectric environment was improved. Therefore, plasmonic devices based on the re-shaped COP mold exhibited a high responsivity to a refractive index of 906 nm/RIU at a wavelength of 625 nm.

Enzyme-responsive fluorescent nanoemulsion based on lipophilic dye liquid.

An enzyme-responsive fluorescent nanoemulsion (NE) based on lipophilic dye liquid (LDL) was developed for alkaline phosphatase (ALP). The response mechanism of the NE involved enzymatic reactions and simultaneous extraction of anions. The LDL-based NE exhibited 3.8 times higher sensitivity than plasticizer-based conventional NE. Detection limit and response range were 2.7 (U L-1) and 5-50 (U L-1), respectively. The response time was reduced to less than half that of the LDL-based membrane.

Fractionation of Single-stranded DNAs with/without Stable Preorganized Structures Using Capillary Sieving Electrophoresis for Aptamer Selection.

Aptamers, single-stranded DNAs/RNAs with a strong and specific interaction towards a target molecule, have wide applications in the fields of medicine and biosensors. In conventional aptamer selection methods, it is difficult to obtain "preorganized" and/or "induced-fit" type of aptamers selectively. In this study, separation and fractionation of single-stranded DNAs with/without stable preorganized structures were carried out using capillary sieving electrophoresis. The fractionated DNAs showed different mobilities and thermodynamic stabilities of their secondary structures; this outcome is deemed to be necessary for the synthesis of novel aptasensors with a desirable sensing mechanism.

Single-step Trypsin Inhibitor Assay on a Microchannel Array Device Immobilizing Enzymes and Fluorescent Substrates by Inkjet Printing.

In this paper, we report a single-step trypsin inhibitor assay on a microchannel array device immobilizing enzymes and substrates by inkjet printing. The microdevice is composed of a poly(dimethylsiloxane) (PDMS) microchannel array that immobilizes trypsin and fluorescent substrates as reactive reagents at the two bottom corners of a microchannel. Inkjet printers allow simple, accurate, and position-selective immobilization of reagents as nanoliter spots. Therefore, plural reactive reagents, such as enzymes and substrates, can be separately immobilized at different positions in the same microchannel without mixing, and thus allowing for single-step operation by simply introducing a sample solution through capillary action. Furthermore, reproducible fabrication and mass production of the device could be expected. In this study, the efficiency of an acidic solution as a spotting agent for protease immobilization to prevent decrease in the fluorescence intensity was confirmed. Additionally, single-step trypsin inhibitor screening was performed using three inhibitors. Finally, we investigated the storage stability of the device and confirmed that it remained stable for at least 10 days.

Direct Measurement of Initial Rate of Enzyme Reaction by Electrokinetic Filtration Using a Hydrogel-plugged Capillary Device.

A novel electrokinetic filtration device using a plugged hydrogel was developed to directly measure the initial rate of enzyme reactions. In the proposed method, the enzyme reaction proceeded only for a short time when the substrate was passed through a thin layer of enzyme trapped by the hydrogel without any lag times for mixing and detection. In experimental conditions, alkaline phosphatase (enzyme) was filtrated at a cathodic-side interface of the plugged hydrogel by molecular sieving effect, providing the thin enzyme zone whose thickness was approximately 100 µm. When 4-methylumberiferyl phosphate (substrate) was electrokinetically introduced into the device after trapping the enzyme, 4-methylumberiferone (product) was generated by the enzyme reaction for only 1.26 s as the substrate passed through the trapped enzyme zone. As a result, the initial rate of the enzyme reaction could be directly calculated to 31.0 µM/s by simply dividing the concentration of the product by the tunable reaction time. Compared to the initial rate obtained by mixing the enzyme and substrate solutions, the value of the maximum velocity of the enzyme reaction was 30-fold larger than that in the mixing method due to the preconcentration of the enzyme by trapping. The Michaelis-Menten constant in the proposed method was 2.7-fold larger than that in the mixing method, suggesting the variation of changes in the equilibrium of complex formation under the experimental conditions.

Lipophilic Fluorescent Dye Liquids: Förster Resonance Energy Transfer-Based Fluorescence Amplification for Ion Selective Optical Sensors Based on a Solvent Polymeric Membrane.

Optical sensors based on solvent polymeric membranes have the potential to measure analytes present in an aqueous solution through the development of a tailored method for a specific target. However, limits in the concentrations of the component dyes have prevented improvements in sensitivity. We propose a Förster resonance energy transfer (FRET)-based fluorescence amplification system for ion-selective optical sensors using a highly fluorescent liquid material composed of a lipophilic phosphonium cation and a pyrene modifying sulfonate anion ([P66614][HP-SO3]), as both the plasticizer and donor, in addition to a combination of the lipophilic phosphonium cation and the fluorescein dodecyl ester anion ([P66614][12-FL]) as the fluorescent sensing dye acceptor. For ion extraction-based sensing, the donor and acceptor were retained in the plasticized PVC membrane with negligible leaching upon exposure to acidic and basic aqueous solutions. Systematic investigation of the donor and acceptor ratios clarified the effect of the amplification factor and the sensitivity of the sensor. At an acceptor doping level of 0.5 mol % (vs donor), an approximately 22-fold higher sensitivity was obtained compared to that of a conventional PVC membrane optical sensor. During ion measurement based on the coextraction of protons and anions, selectivity following the Hofmeister order was observed, which was controlled by the addition of ionophores. The proposed FRET system based on a lipophilic fluorescent liquid material has the potential to significantly improve the sensitivities of optical sensors using solvent polymeric membranes with high selectivities for various target analytes.

Highly sensitive optical ion sensor with ionic liquid-based colorimetric membrane/photonic crystal hybrid structure.

An ionic liquid-based thin (~ 1 µm) colorimetric membrane (CM) is a key nano-tool for optical ion sensing, and a two-dimensional photonic crystal slab (PCS) is an important nano-platform for ultimate light control. For highly sensitive optical ion sensing, this report proposes a hybrid of these two optical nano-elements, namely, a CM/PCS hybrid. This structure was successfully fabricated by a simple and rapid process using nanoimprinting and spin-coating, which enabled control of the CM thickness. Optical characterization of the hybrid structure was conducted by optical measurement and simulation of the reflection spectrum, indicating that the light confined in the holes of the PCS was drastically absorbed by the CM when the spectrum overlapped with the absorption spectrum of the CM. This optical property obtained by the hybridization of CM and PCS enabled drastic improvement in the absorption sensitivity in Ca ion sensing, by ca. 78 times compared to that without PCS. Experimental and simulated investigation of the relation between the CM thickness and absorption sensitivity enhancement suggested that the controlled light in the PCS enhanced the absorption cross-section of the dye molecules within the CM based on the enhanced local density of states. This highly sensitive optical ion sensor is expected to be applied for micro-scale bio-analysis like cell-dynamics based on reflectometric Ca ion detection.

A lipophilic ionic liquid-based dye for anion optodes: importance of dye lipophilicity and application to heparin measurement.

Herein, a fully lipophilic ionic liquid (IL) comprising a lipophilic fluorescein anion and a trihexyltetradecylphosphonium cation was synthesized and used as the plasticizer for a plasticized poly(vinyl chloride) (PVC) membrane optode. Systematic investigation of the alkyl chain length of the fluorescein anion proved the significance of lipophilicity for obtaining the reversible absorbance measurements. A PVC membrane fabricated with the synthesized lipophilic IL was observed to comprise an unusually high dye concentration (915 mmol kg-1) and exhibited good sensitivity as well as response time in its sensor performance. The sensitivity of the presented PVC membrane was 26-fold higher than that of a conventional optode membrane with the same membrane thickness and the same lipophilic dye of typical dye content (1 wt%). The response time was observed to be >120-fold faster by using a significantly thinner PVC membrane (approx. 140 nm). Heparin is known to be a polyanionic anticoagulant, and the presented PVC membrane exhibited an extremely fast response (20-150 seconds) to the heparin in diluted serum within the required concentration region. Thus, the lipophilic IL-based dye could significantly improve the sensor performance in conventional optodes, especially for an analyte showing slow diffusion, such as macromolecular heparin.