Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance.

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids.

Construction of a fusion protein consisting of α-1,3-glucan-binding domains and tetrameric red fluorescent protein, which is involved in the aggregation of α-1,3-glucan and inhibition of fungal biofilm formation.

Agl-KA, an α-1,3-glucan-hydrolyzing enzyme from Bacillus circulans KA-304, has three α-1,3-glucan-binding domains DS1, CB6, and DS2 (DCD). While their individual binding activities toward insoluble α-1,3-glucan and fungal cell-wall are weak, the three domains in combination bind strongly to the α-1,3-glucan and the cell-wall. In this study, we constructed DCD-tetraRFP by fusing DCD with DsRed-Express2, a tetrameric red fluorescent protein. DCD-tetraRFP forms a tetramer in an aqueous solution and contains twelve substrate-binding domains in one complex. We also constructed DCD-monoGFP by fusing DCD with AcGFP1, a monomeric green fluorescent protein. The molecular weight of DCD-tetraRFP and DCD-monoGFP were compared. The results of gel filtration chromatography and dynamic light scattering indicated that DCD-tetraRFP was larger than DCD-monoGFP, suggesting that DCD-tetraRFP had a tetrameric structure. In addition, DCD-tetraRFP bound to insoluble α-1,3-glucan strongly, and the amount of DCD-tetraRFP binding to 0.01% α-1,3-glucan was about twice of DCD-monoGFP. The Kd values of DCD-tetraRFP (measurements per subunit) and DCD-monoGFP were 0.16 and 0.84 µM, respectively. Adding DCD-tetraRFP to a suspension of α-1,3-glucan caused glucan aggregation; however, adding DCD-monoGFP did not. These data suggested that DCD-tetraRFP had four DCDs sterically arranged in different directions so that DCD-tetraRFP cross-linked with the substrate, causing aggregation. Lastly, the aggregates of DCD-tetraRFP and α-1,3-glucan captured Aspergillus oryzae conidia and decreased their biofilm formation by 80% in a 24-well dish.

Pocketable Biosensor Based on Quartz-Crystal Microbalance and Its Application to DNA Detection.

Quartz-crystal microbalance (QCM) is a technique that can measure nanogram-order masses. When a receptor is immobilized on the sensor surface of a QCM device, the device can detect chemical molecules captured by the mass change. Although QCM devices have been applied to biosensors that detect biomolecules without labels for biomolecular interaction analysis, most highly sensitive QCM devices are benchtop devices. We considered the fabrication of an IC card-sized QCM device that is both portable and battery-powered. Its miniaturization was achieved by repurposing electronic components and film batteries from smartphones and wearable devices. To demonstrate the applicability of the card-sized QCM device as a biosensor, DNA-detection experiments were performed. The card-sized QCM device could detect specific 10-mer DNA chains while discerning single-base differences with a sensitivity similar to that of a conventional benchtop device. The card-sized QCM device can be used in laboratories and in various other fields as a mass sensor.

Mixed Polyplex Micelles with Thermoresponsive and Lysine-Based Zwitterionic Shells Derived from Two Poly(vinyl amine)-Based Block Copolymers.

Two series of poly(vinyl amine) (PVAm)-based block copolymers with zwitterionic and thermoresponsive segments were synthesized by the reversible addition-fragmentation chain transfer polymerization. A mixture of the two copolymers, poly(N-acryloyl-l-lysine) (PALysOH) and poly(N-isopropylacrylamide) (PNIPAM), which have the same cationic PVAm chain but different shell-forming segments, were used to prepare mixed polyplex micelles with DNA. Both PVAm-b-PALysOH and PVAm-b-PNIPAM showed low cytotoxicity, with characteristic assembled structures and stimuli-responsive properties. The cationic PVAm segment in both block copolymers showed site-specific interactions with DNA, which were evaluated by dynamic light scattering, zeta potential, circular dichroism, agarose gel electrophoresis, atomic force microscopy, and transmission electron microscopy measurements. The PVAm-b-PNIPAM/DNA polyplexes showed the characteristic temperature-induced formation of assembled structures in which the polyplex size, surface charge, chiroptical property of DNA, and polymer-DNA binding were governed by the nitrogen/phosphate (N/P) ratio. The DNA binding strength and colloidal stability of the PVAm-b-PALysOH/DNA polyplexes could be tuned by introducing an appropriate amount of zwitterionic PALysOH functionality, while maintaining the polyplex size, surface charge, and chiroptical property, regardless of the N/P ratio. The mixed polyplex micelles showed temperature-induced stability originating from the hydrophobic (dehydrated) PNIPAM chains upon heating, and remarkable stability under salty conditions owing to the presence of the zwitterionic PALysOH chain on the polyplex surface.

Constructive Optimization of a Multienzymatic Film Based on a Cascade Reaction for Electrochemical Biosensors.

The application of a multienzyme cascade reaction in electrochemical biosensors has the advantage of expanding the target substrates in addition to selectivity combining multiple enzymes on an electrode. However, the multienzyme system has the drawback of inefficient substance conversion because of the time-consuming passing of intermediates between the enzymes and/or diffusional loss of the intermediates. In this study, the optimal construction of a multienzymatic film in an ammonia detection sensor was investigated using a cascade reaction of l-glutamate oxidase and l-glutamate dehydrogenase as a model sensor. Three enzymatic films were prepared: (1) a mixed film designed to have a short diffusional distance between closely located enzymes, (2) a normal-sequential layered film arranged for the correct reaction pathway, and (3) a reverse-sequential layered film as a negative control. This was followed by comparison of the conversion efficiency of ammonia to hydrogen peroxide using time-dependent potentiometric measurements of a Prussian blue electrode determining the hydrogen peroxide amount. The results indicate that the conversion efficiency of the normal-sequential layered film was the highest among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction showed that compared to the mixed film (34%), a higher conversion efficiency of 92% was obtained in the first enzymatic reaction step. These findings will promote the use of multienzymatic cascade reaction systems not only in biosensors and bioreactors but also in various industrial fields.

Printed Organic Transistor-based Biosensors for Non-invasive Sweat Analysis.

This review describes recent advances in biosensors for non-invasive human healthcare applications, especially focusing on sweat analysis, along with approaches for fabricating these biosensors based on printed electronics technology. Human sweat contains various kinds of biomarkers. The relationship between a trace amount of sweat biomarkers partially partitioned from blood and diseases has been investigated by omic analysis. Recent progress in wearable or portable biosensors has enabled periodic or continuous monitoring of some sweat biomarkers while supporting the results of the omic analysis. In this review, we particularly focused on a transistor-based biosensor that is highly sensitive in quantitatively detecting the low level of sweat biomarkers. Furthermore, we showed a new approach of flexible hybrid electronics that has been applied to advanced sweat biosensors to realize fully integrated biosensing systems wirelessly connected to a networked IoT system. These technologies are based on uniquely advanced printing techniques that will facilitate mass fabrication of high-performance biosensors at low cost for future smart healthcare.

Entropy-Driven Mechanisms between Disulfide-Bond Formation Protein A (DsbA) and B (DsbB) in Escherichia coli.

A disulfide-bond formation system for nascent proteins in the Escherichia coli periplasm contains efficient electron transfer systems for the catalysis of oxidation. This electrochemical system has interesting implications in vivo. Disulfide bonds are formed by disulfide-bond formation protein A (DsbA), which contains two reactive cysteines. DsbA is reoxidized by a membrane protein, disulfide-bond formation protein B (DsbB), which has four catalytic cysteines. The oxidation of DsbA by DsbB seems energetically unfavorable on the basis of the redox potential. The oxidizing power of ubiquinone (UQ), which endogenously binds with DsbB, is believed to promote this reaction. However, using UQ-deficient DsbB, it was found that the oxidation of DsbA by DsbB proceeds independently of UQ. Thus, the reaction mechanism of DsbA oxidation by DsbB is under debate. In this study, we used the quartz crystal microbalance technique, which detects the intermediate complex between DsbA and DsbB during DsbA oxidation as a change in mass, to obtain kinetic parameters of DsbA oxidation under both the oxidized and reduced states of UQ at acidic and basic pH. In addition, we utilized sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility shift assay technique to determine the pK a of the cysteine thiol groups in DsbA and DsbB. We found that DsbA oxidation proceeded independently of UQ and was greatly affected in kinetics by the shuffling of electrons among the four cysteine residues in DsbB, regardless of pH. These results suggest that DsbA oxidation is driven in an entropy-dependent manner, in which the electron-delocalized intermediate complex is stabilized by preventing a reverse reaction. These findings could contribute to the design of bio-inspired electrochemical systems for industrial applications.

Noninvasive Sweat-Lactate Biosensor Emplsoying a Hydrogel-Based Touch Pad.

This study is the first report demonstrating proof-of-concept for a hydrogel-based touch sensor pad used for the non-invasive extraction and detection of sweat components. The sensor device was composed of an electrochemical L-lactate biosensor covered with an agarose gel in a phosphate buffer saline. When human skin contacts the agarose gel, L-lactate in sweat was continuously extracted into the gel, followed by in-situ potentiometric detection without controlled conditions. This novel type of sweat sensor is expected to enable the simple, non-invasive daily periodic monitoring of sweat biomarkers for advanced personal healthcare methods in the future.

Synthesis, Assembled Structures, and DNA Complexation of Thermoresponsive Lysine-Based Zwitterionic and Cationic Block Copolymers.

A series of anionic, zwitterionic, and cationic lysine-based block copolymers with a thermoresponsive segment were synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization of N-acryloyl- N-carbobenzoxy-l-lysine [A-Lys(Cbz)-OH], which contains a carboxylic acid and a protected amine-functionality in the monomer unit. Carboxylic acid-containing homopolymers, poly(A-Lys(Cbz)-OH), with predetermined molecular weights with relatively low polydispersities were initially synthesized by RAFT polymerization of A-Lys(Cbz)-OH. The chain extension of the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) to N-isopropylacrylamide (NIPAM) via the RAFT process and subsequent deprotection afforded the zwitterionic block copolymer composed of thermoresponsive poly(NIPAM) and poly(A-Lys-OH), which exhibited switchability among the zwitterionic, anionic, and cationic states by pH change. The assembled structures and thermoresponsive and chiroptical properties of these block copolymers were evaluated by dynamic light scattering, circular dichroism, and turbidity measurements. Finally, the cationic block copolymer, poly(A-Lys-OMe)- b-poly(NIPAM), was obtained by the methylation of the carboxylic acid group in the zwitterionic poly(A-Lys-OH) segment. Selective interactions of DNA with the cationic poly(A-Lys-OMe) segment in the lysine-based block copolymer were further evaluated by agarose gel electrophoresis and atomic force microscopy measurements, which revealed characteristic assembled structures and temperature-responsive properties of the polyplexes.

Probing Multiple Binding Modes of DNA Hybridization: A Comparison between Single-Molecule Observations and Ensemble Measurements.

Interactions between biomolecules are generally analyzed by ensemble measurements, assuming that the interactions occur in a single binding manner. However, such interactions may occur via multiple binding modes. We investigated the kinetics of DNA hybridization as a multiple dynamic model of biomolecular interactions. Two kinetic analyses were performed with a single-molecule observation using total internal reflection fluorescence microscopy (TIRFM) and with ensemble measurements using a quartz-crystal microbalance (QCM) biosensor. We observed the DNA hybridization of 8 and 12 bp DNAs with random sequences and dA12-dT12 and calculated the kinetic parameters, including the dissociation rate constant (koff). Hybridization of 8 bp DNA proceeded mainly via a single binding mode. However, hybridization of 12 bp DNA indicated at least two different binding modes and dA12-dT12 hybridization showed multiple binding modes. For the multiple binding interactions, the kinetic parameters obtained from TIRFM and QCM were different because kinetic parameters obtained from QCM indicate average number of molecules, whereas those from TIRFM indicate average association time. The present study revealed the details of multiple interactions, which can be utilized for better understanding of not only DNA hybridization but also biomolecular interaction mechanisms.

Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance.

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (kon, koff, and Ka, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA30·dT30 and dG30·dC30) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA30·dT30 and dG30·dC30, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG30·dC30 with high Ka and small koff values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low kon and koff values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high Ka values owing to the high kon values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.

In situ monitoring of structural changes during formation of 30S translation initiation complex by energy dissipation measurement using 27-MHz quartz-crystal microbalance.

Ribosome is a bionanomachine that facilitates an orderly translation process during protein synthesis in living cells. Real-time monitoring of conformational changes in ribosomal subunits in aqueous solution is important to understand the regulatory mechanism of protein synthesis, because conformational changes in ribosome in E. coli have been predicted to operate the switch from translation initiation to an elongation process during translation. We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitoring of conformational changes in pre-30S translation initiation complex in response to the binding of fMet-tRNA(fMet) in aqueous solution. The addition of fMet-tRNA(fMet) caused changes in the physical property (increased dehydration and elasticity) in 30S ribosomal subunit in the presence of mRNA and IF2/guanosine 5'-triphosphate (GTP) on the QCM plate. Furthermore, two sequential changes triggered by the addition of fMet-tRNA(fMet) were observed in 30S ribosomal subunit bound to mRNA in the presence of IF2/GTP and IF3. These observations suggest that the structural changes in 30S ribosomal subunit caused by the binding of fMet-tRNA(fMet) with IF2/GTP in the presence of IF3 could act as a switch to regulate the orderly processing in the construction of translation initiation complex, because the structural distinction can be a guidepost in the process for the relevant biomolecules.

Arginine arrangement of bacteriophage λ N-peptide plays a role as a core motif in GNRA tetraloop RNA binding.

A simple α-helical N-model-peptide was designed to investigate the role of the arginine-rich motif of bacteriophage λ N-peptide in selective binding with boxB RNA. The five-arginine arrangement of native N-peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random-nucleotide region) was carried out with N-model-peptide immobilized on a 27 MHz quartz-crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (Ka =10(7) -10(8) M(-1) ). Many selected RNAs contained GNR(N)A-type loops (similar to the boxB RNA motif recognized by the native N-peptide). Fragments and minimal RNAs containing the GNRA-type loop also bound to N-model-peptide (Ka =10(6) -10(7) M(-1) ). The RNA recognition specificity of the peptide was studied by changing the "closing" U-A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N-model-peptide). It was concluded that the five-arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N-peptide adds the recognition ability for a loop-RNA sequence.

Real-time monitoring of intermediates reveals the reaction pathway in the thiol-disulfide exchange between disulfide bond formation protein A (DsbA) and B (DsbB) on a membrane-immobilized quartz crystal microbalance (QCM) system.

Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.

Translation enhancer improves the ribosome liberation from translation initiation.

For translation initiation in bacteria, the Shine-Dalgarno (SD) and anti-SD sequence of the 30S subunit play key roles for specific interactions between ribosomes and mRNAs to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein S1, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown. In this paper, we investigated the effects of the SD and enhancer sequences on the binding kinetics of the 30S ribosomal subunits to mRNAs and their translation efficiencies. mRNAs with both the SD and translation enhancers promoted the amount of protein synthesis but destabilized the interaction between the 30S subunit and mRNA by increasing the dissociation rate constant (koff) of the 30S subunit. Based on a model for kinetic parameters, a 16-fold translation efficiency could be achieved by introducing a tandem repeat of adenine sequences (A20) between the SD and translation enhancer sequences. Considering the results of this study, translation enhancers with an SD sequence regulate ribosomal liberation from translation initiation to determine the translation efficiency of the downstream coding region.

In situ monitoring of a trace intermediate during DNA phosphorylation by T4 polynucleotide kinase for transient kinetic studies.

Formation and decomposition of the enzyme-substrate (ES) complex during phosphorylation by T4 polynucleotide kinase (T4 PNK) of dsDNAs were monitored using a highly sensitive quartz crystal microbalance (QCM) to determine kinetic parameters, which were characterised in comparison with those of other enzymes such as DNA polymerase and exo- and endo-nucleases.

Small mass-change detectable quartz crystal microbalance and its application to enzymatic one-base elongation on DNA.

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.

Real-time monitoring of cell-free translation on a quartz-crystal microbalance.

The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fMet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.

Added mass effect on immobilizations of proteins on a 27 MHz quartz crystal microbalance in aqueous solution.

During the immobilization process of proteins onto an Au-surface of a 27 MHz quartz crystal microbalance (QCM) in aqueous solutions, apparent large frequency changes (DeltaF(water)) were observed compared with those in the air phase (DeltaF(air)) due to the interaction with surrounding water of proteins. On the basis of an energy-transfer model for the QCM, the apparent added mass in the aqueous solution [(-DeltaF(water))/(-DeltaF(air)) - 1] could be explained by frictional forces at the interface of proteins with aqueous solutions. When [(-DeltaF(water))/(-DeltaF(air)) - 1] values for various proteins were plotted against values relating to the friction (antimobility), such as values of the molecular weight divided by the sedimentation coefficient (Mw/s), the inverse of the diffusion coefficient (1/D), and the volume divided by the surface area (volume/surface area = apparent radius) of proteins, there were good linear correlations. Thus, observations of the larger DeltaF(water) than DeltaF(air) for protein immobilizations on the QCM can be simply explained by the friction effect at the interface between proteins and the aqueous solution. Thus, QCM would be a mass sensor based on mechanical oscillation motion even in aqueous solutions.

In situ monitoring of conformational changes of and peptide bindings to calmodulin on a 27 MHz quartz-crystal microbalance.

Conformational changes of calmodulin (CaM) by additions of Ca(2+) ions and bindings of CaM-binding peptides to Ca(2+)/CaM followed by conformational changes were monitored by a CaM-immobilized 27 MHz quartz-crystal microbalance (QCM) with an admittance analysis. Both the binding and the conformational change events could be detected from the time-dependence of frequency decreases (mass increases) and energy dissipation decreases (elasticity increases), respectively. When Ca(2+) ions were injected to a QCM cell on which biotinylated CaM was immobilized with avidin-biotin interactions, a frequency increase (a mass decrease) and an energy dissipation decrease (an elasticity increase) were observed because of the dehydration and the elasticity increase caused by conformational changes from the flexible CaM to the rigid Ca(2+)/CaM exposing the hydrophobic surface. In the case of the addition of CaMKII-peptide in the Ca(2+)/CaM-immobilized QCM, the immediate frequency decrease (the mass increase) due to the binding of the peptide to Ca(2+)/CaM and the following energy dissipation decrease (the elasticity increase) with a time lag were observed. This suggests that the interaction of the CaMKII-peptide to Ca(2+)/CaM follows an allosteric binding mode. Binding kinetics of the peptide to Ca(2+)/CaM (k(1) and k(-1)) and kinetics of the following conformational change of Ca(2+)/CaM (k(2) and k(-2)) could be obtained. This technique is useful to investigate biomolecular interactions involving the conformational and/or viscoelastic property changes that are biologically important.