Proteins possess sophisticated functions in order to maintain strict life phenomenon. Therefore, the molecules have an aspect as fine materials, as in the example of application tools to signal amplification and molecular recognition in analytical chemistry. On the other hand, many artificial materials have unique functions, which are different from that of biomolecules. Thus, the complex formation between proteins and artificial materials has a great potential to create attractive materials. We have developed novel hybrid materials composed of proteins, mainly enzymes, and inorganic nanosheets. In this review, hybrid materials, reported by our group, are summarized while focusing on applications to analytical chemistry.
Although the lock-and-key principle is a basic concept not only for highly selective enzymatic equilibria/reactions, but also for molecular recognition and supramolecular assembly, there are some problems in applying the principle to chemical sensing with artificial chemosensors. One of the most inherent examples may be an unexpected solvation, i.e., mutual entropy losses, based on an enthalpy-entropy compensatory relationship. So far, we have proposed a novel sensing methodology, “supramolecular allosteric signal-amplification sensing” (SASS), enabling us to selectively and sensitively detect various analytes that are difficult to sense in using lock-and-key-type chemosensors. In this review, we want to highlight the concept, strategy, and applications of SASS with our recent studies.
We developed carbon film-based electrodes by using an unbalanced magnetron (UBM) and an electron cyclotron resonance (ECR) sputtering equipment, and applied for the electroanalysis of four kinds of heavy metal ions: cadmium (Cd2+), lead (Pb2+), arsenic (As3+) and selenium (Se4+). Au nanoparticle embedded carbon (AuNP-C) film was formed by UBM co-sputtering. Regarding the detection of cadmium (Cd2+) and lead (Pb2+) ions, anodic stripping voltammetry measurement was performed with our carbon thin film electrode. The detection limits of Cd2+ and Pb2+ were 0.25 ppb and 1.0 ppb on our electrode. These values were better than those on the conventional GC electrode, also sufficient for the environmental analysis. On the other hand, the AuNP-C electrode was used for the detection of arsenic ions (As3+), which cannot be preconcentrated on the pure carbon electrode. A detection limit of 0.55 ppb was achieved, which is a sufficient performance for the environmental analysis. Reproducible results could be obtained for an As3+ measurement after Au was further electrodeposited on AuNPs because AuNPs are embedded in the carbon film, which prevented the AuNPs detaching from the electrode surface. In the case of selenium ion (Se4+) detection, an electrode in which Au was electrodeposited only on AuNPs at AuNP-C (Au/AuNP-C) electrodes was used because of the slow deposition rate of Se4+. Improved sensitivity and low detection limit were achieved compared with the electrode before the electrodeposition.
Molecularly imprinted polymers (MIPs) are attracting attention as artificial molecular recognition materials instead of unstable natural receptors. Previously, we had synthesized MIPs toward Bisphenol A (BPA). The prepared MIPs could capture not only BPA but also 17β-estradiol, an estrogen steroid hormone. This suggests that the prepared MIPs have binding properties similar to estrogen receptors. In this study, the binding properties of structurally related compounds toward the MIPs were investigated by in silico analysis focusing on the three-dimensional structure of the compounds. These investigations revealed that various factors obtained from each global minimum structure of compounds, such as the positions of interacting groups, conformational flexibility, van der Waals volume, intra-atomic distance between interacting groups on the compounds and pKa of interacting group, are related to experimental retention factors. Using molecular descriptors calculated from the compounds, four types of prediction models for retention factors were derived by a quantitative structure-property relationship analysis, and the calculated values and experimental values showed a high correlation. A consensus pharmacophore was extracted from 7 compounds with strong affinity to the MIPs. These models could be expected to be used for predicting the retention factor from global minimum structures of compounds and for pre-screening candidate compounds before starting actual binding experiments. In addition, it could potentially be used to predict and design compounds with strong affinity for MIPs by a reverse analysis method.
In this work, an inkjet-printed single-step and homogeneous bioassay microdevice was developed with a poly(dimethylsiloxane) (PDMS) microchannel for easy fabrication and mass production. An inkjet printer can dispense reactive reagents as nanoliter droplets on a PDMS microchannel accurately. Therefore, homogeneous and reproducible dispensing of reagents can be achieved. In a previous report, a complicated fabrication procedure for combining two PDMS microchannel arrays immobilizing two reactive reagents was required. Therefore, in the present report, a new immobilizing method, which involves the patterning of two reactive reagents on two bottom corners of a PDMS microchannel as reagent spots separately based on inkjet printing, was developed. Based on this method, we developed single-step and homogeneous bioassay microdevice that patterns a biotin-sulfonic acid group-introduced graphene oxide conjugate and a fluorescently labeled streptavidin with the same microchannel. For a homogeneous bioassay, the dissolution of immobilized reactive reagents by sample introduction is essential. Therefore, the immobilization matrix, which was mixed with reactive reagents, was investigated. We found that trehalose exhibited favorable characteristics concerning rapid dissolution and uniformity. Then, a single-step detection of biotin was carried out. The detection limit was 0.68 ng mL−1, which was approximately 600-times lower than that of previously reported method. Furthermore, biotin selectivity based on a bioassay microdevice was confirmed. In the future, an immobilization method based on inkjet printing can be applied to various bioassay devices based on the microchannel.
We have developed a dimethylacrylamide (DMAA) coplymerized optical glucose sensing film with a semi-interpenetrating structure in which the bisbenzoboroxol (bisBB) receptor–dihydroxyazobenzene (DHAB) dye complex serves as a cross-linking point of the polymer. The color change response of the sensing film to glucose is amplified by swelling of the polymer, as the receptor–dye complex at the cross-linking point of the film dissociates due to competitive complexation of the receptor with glucose. It was found that the glucose response of the sensing film was strongly dependent on the film composition. Of the eight sensing films with different compositions we prepared, a film containing 0.5 mol % of bisBB receptor and 0.14 mol % of DHAB dye with respect to DMAA showed the largest reversible glucose response within the physiological concentration range.
We synthesized a bis-benzoboroxole-type glucose receptor (bis-BB-AN) in which the glucose recognition site of the bis-phenylboronic acid receptor with anthracene fluorophore (bis-PBA-AN), which is known as an excellent glucose receptor, was replaced with benzoboroxole (BB). The glucose recognition abilities of these bis-PBA-AN and bis-BB-AN receptors were evaluated on the basis of the alizarin red S (ARS) colorimetric assay and the fluorescence assay. The complex formation constant with glucose estimated by the ARS colorimetric assay was 0.6 mol−1 L for the bis-PBA-AN receptor, and 30 mol−1 L for the bis-BB-AN receptor, respectively: The large complex formation constant of the bis-BB-AN receptor compared to the bis-PBA-AN receptor reflects the higher glucose recognition ability of BB than that of PBA. However, the complex formation constant with glucose estimated by the fluorescence assay was 3600 mol−1 L for bis-PBA-AN and 170 mol−1 L for bis-BB-AN: The bis-PBA-AN receptor showed an ~20- times greater glucose recognition ability than the bis-BB-AN receptor. These results suggest that the excellent glucose recognition ability of bis-PBA-AN is due to the glucose-specific fluorescence switching mechanism during weak binding, rather than to the ability to form a stable complex with glucose.
Thiazole orange (TO), a typical monomethine cyanine dye, does work as a useful fluorogenic indicator in FID (fluorescent indicator displacement) assay for targeting HIV-1 TAR (trans-activation responsive region) RNA. A comparison of the binding and signaling functions of a series of monomethine cyanine dyes, including TO-PRO1, YO-PRO-1 JO-PRO-1 and BO, reveals that TO is the best indicator for TAR RNA among these dyes. The binding of TO is highly selective to TAR RNA over duplex RNA, and the dissociation constant, Kd, reaches 60±7 nM (pH 6.5, 10 mM sodium phosphate, 50 mM NaCl, 0.1 mM EDTA, 5°C). The binding to TAR RNA is accompanied by a significant light-up response (ϕfree < 0.01, ϕbound = 0.198), where the light-up factor, I/I0 (I and I0 denote the fluorescence intensities in the presence and absence of the target RNA, respectively), reaches ca. 370-fold (λex = 501.5 nm, λem = 530.4 nm). In addition, an examination of the Z'-factor in a 384-well microplate format resulted in a value of > 0.5, revealing the high quality of the TO-based assay for HTS (high throughput screening). These promising functions of TO are discussed as a rational basis for the advanced design of RNA-binding indicators for FID assay that targets HIV-1 TAR RNA.
With the aim of developing a subcutaneously implantable optical sugar sensor that can continuously measure blood glucose levels, we have developed a novel dye that changes the absorption spectrum in the near-infrared wavelength region by forming a complex with glucose receptors. As a result of synthesizing six types of azaBODIPY dyes having a catechol moiety, it was found that the absorption spectrum of each dye changes due to the complex formation with phenylboronic acid (PBA), which is a model compound of sugar receptors. Among the receptor-sensitive dyes we synthesized, thienyl-azaBODIPY, in which three thienyl groups were introduced into the molecule, showed the maximum absorption wavelength (732 nm) on the longest wavelength side. It was found that when glucose was added to a solution containing thienyl-azaBODIPY and the glucose receptor bisbenzoboroxol (bisBB), the absorbance in the near-infrared wavelength region changed significantly depending on the glucose concentration.
The quantification of electrolytes in biological fluids plays an important role for evaluating the human health condition. This work proposes a disposable and simple microfluidic paper-based analytical device (μPAD) for the quantitative colorimetric detection of salivary Na+, K+ and Ca2+, relying on ion-selective optodes (ISOs). Nanoscale ISOs composed of pluronic F-127 surfactant-based micelles were used as a colorimetric indicator system, reducing the assay time compared to the previously reported bulk membrane approach. Devices consist of three functionalized paper layers assembled by lamination, with the optical signal read out from the bottom layer. Saliva was chosen as the biological specimen because it is painlessly collected and easy-to-handle, which makes it suitable for POC assays. Simultaneous Na+, K+ and Ca2+ detection by a single μPAD was successfully confirmed by using aqueous metal ion samples with various concentrations of Na+, K+ and Ca2+ within the biologically relevant range. The simultaneous detection of the three cationic species enabled the correction of the three ion quantities by eliminating the interference from other ions using the calculated selectivity coefficients. For proof-of-concept, Na+ quantification in a human saliva sample was conducted by the developed μPADs. The calibration curve for this assay was prepared by applying artificial saliva to the μPADs. The concentrations of Na+ in spiked human saliva were also measured by the μPADs. The obtained recovery rates were 78-132 %, which indicates the quantification of the added Na+ in the real sample matrix. Finally, this μPAD was successfully integrated into a smartphone-based assay, which enables salivary electrolyte quantification in resource poor settings. The developed μPADs satisfy the criteria of low-cost (< $0.3/device), rapidity (< 5 min) and simple analytical procedure compared to the conventional ion-selective electrode (ISE) method.
Urinalysis is widely used in routine medical checkups. Conventionally, colorimetric paper strips currently available on the market are applied. Although they allow to rapidly obtain results, their use is often not straightforward, because a timed readout is required, followed by comparison with a color chart. D-μPADs (distance-based microfluidic paper-based analytical devices) have gained attention for enabling highly intuitive signal readout. During liquid flow through a paperfluidic channel, target analytes are sequentially depleted from the sample through reaction with colorimetric indicators and/or adsorption to the paper surface, so that the length of color-changed sections correspond to the analyte content. Therefore, D-μPADs are expected to solve the problems of existing urinalysis tests. The final goal is the development of D-μPADs for the simultaneous detection of urinary protein (albumin), glucose and hemoglobin. Colorimetric distance-based calibration curves were acquired for all analytes in artificial urine matrix containing electrolytes and organic compounds. For quantitative assays, evaluation of color-changed distances was conducted by computer software. The first cases of distance-based albumin, glucose and hemoglobin readouts in relatively complex artificial urine matrix are reported. For albumin detection, the color uniformity of the colorimetric indicator was improved by paper surface modification with poly(4-styrenesulfonic acid), an anionic polymer. For glucose detection, signal visibility was significantly improved by paper surface modification with chitosan, a biopolymer that works as immobilization support for enzymes. Finally, we evaluated the effect of urinary components on the assay results. It was confirmed that D-μPADs for the hemoglobin assay require a treatment to reduce the effect of albumin adsorption on the paper substrate.
Methylene blue is widely utilized as an indicator and dye in bioassays. For the simultaneous detection of many samples in bioassays, a multiple assay device is desired. Here, we present an array device based on local redox cycling for the detection of small droplets of methylene blue. The device consists of 8 row, 8 column electrodes connected to interdigitated array (IDA) electrodes at individual crossing points to form 64 sensor points. By segmenting the sensor points with droplets, each point works as an electrochemical cell, thus allowing multi-point detection of the analyte. In this study, methylene blue was successfully detected using redox cycling. Also, multiple detection of methylene blue was achieved. Because methylene blue binds strongly to DNA via intercalation, this interaction will be detected as an electrochemical signal via redox cycling. In particular, the proposed method will be useful for the simultaneous detection of multiple samples in droplet digital PCR.
The proton spin-spin relaxation times (T2) of ion-selective electrode membranes prepared from different solvents were investigated. The potentiometric polymeric membranes based on bis(12-crown-4) as an ionophore for the Na+ ion-selective electrodes were chosen as model ISE membranes. The solvents applied to the preparation of the potentiometric polymeric membranes were tetrahydrofuran (THF), 1,4-dioxane, acetone, methylethylketone (MEK) and chloroform. T2 measurements were performed using the Hahn-Echo, Solid-Echo and Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences. Analyses of the T2 measurements by Hahn-Echo pulse sequences could provide the average T2 values of the whole potentiometric polymeric membranes. The average T2 values will indicate the degree of plasticization for the whole membranes. Moreover, the normalized derivative spectra by T2 measurements of the Solid-Echo and CPMG pulse sequences could visualize the degree of plasticization for such potentiometric polymeric membranes. On the other hand, differences in the solvents were found to affect the electromotive force (EMF) response in the low-concentration region of the Na+ ions and selectivity coefficients for the ISE membranes. These results reveal a relationship of the acquired T2 measurement data with the EMF response and the ion-selectivity for a series of the Na+-ISEs based on bis(12-crown-4). The most plasticized ISE membrane by THF exhibited the best EMF response and ion-selectivity.
Porphyromonas gingivalis, known as an anaerobic gram-negative bacteria, is associated with the progress of a periodontitis. Since gingipain (gp), a specific protease secreted from its bacteria, is its main pathogenicity, gp has been utilized as a marker of the periodontitis. Here, an electrochemical protease assay using a ferrocenylpeptide probes was applied to the detection of gp activity; the probe was cleaved with its protease activity and current peak decreased by the released ferrocene part to balk solution. A series of ferrocenylpeptides as specific substrates of Arg-gp (Rgp) and Lys-gp (Kgp) was synthesized: FRC and FKC having cysteine residue; FRSS and FKSS having lipoic acid; FRpra and FKpra having propargylglycine; FRDE and FRDK having cysteine and D-type amino acid. The electron transfer rate constant of all of ferrocenylpeptide immobilized on a gold electrode were estimated by Lavirons analysis. The obtained electron transfer rate constant were 400-3500 s−1, where fastest one of 3500 s−1 was obtained from FRC immobilized electrode. Peak currents of these electrodes were decreased after the treatment of a sample containing Porphyromonas gingivalis. The detection limits of Porphyromonas gingivalis were 1.0 × 107, 5.0 × 106, 3.3 × 106, 5.0 × 106, and 5.0 × 106 cells, for FRPra- FKPra-, FRC-, FRDE-, and FRDK-immobilized electrodes, respectively. FRDE- and FRDK-immobilized electrodes were applied for patients suffering from periodontitis. It was successfully classified between the patients and healthy peoples using the FRDK-immobilized electrode. These results suggest that gp activity is connected with a clinical condition of periodontitis, and it is expected to be applied to a periodontal disease screening.
We developed lanthanoid-doped upconversion nanoparticles (UCNPs) and attempted to apply them as wavelength conversion devices to near-infrared luminescent sugar sensors. By doping yttrium fluoride with ytterbium and thulium, UCNPs (NaYF4: Yb/Tm) showing upconversion (UC) emission in the near-infrared region (800 nm) were synthesized. A receptor-sensitive near-infrared absorbing dye that increases the absorbance in the near-infrared region (700-800 nm) by complex formation with phenylboronic acid (PBA) as a sugar receptor was immobilized on core-shell type lanthanoid UCNPs covered with a silica layer. When fructose was added to a dye-immobilized core-shell type UCNPs dispersion solution containing a certain amount of PBA (1.0 × 10−3 mol L−1), the UC emission intensity at 800 nm increased with increasing the concentration of fructose (0 mol L−1 to 3.0 × 10−1 mol L−1). This result indicates that the internal filter effect between the dye and UCNPs is eliminated by a change in the absorption spectrum of the immobilized receptor-sensitive dye due to a competitive complex formation reaction accompanying the increase in the fructose concentration. And as a result, the UC emission intensity increased. It was found that when the fructose concentration was 3.0 × 10−1 mol L−1, the UC emission intensity increased by 14.6% compared to the case without fructose.