Wireless-electrodeless quartz-crystal-microbalance biosensors for studying interactions among biomolecules: a review.
It has been well recognized that quantitative evaluation of interactions among bio-molecules and between cell and proteins will achieve significant progress in the protein design and drug discovery. For this purpose, various tag proteins have been developed. However, conventional affinity tag systems include large protein tags such as green-fluorescent proteins (9 ~ > 27kDa), FLAGs with specific antibodies (9 ~ > 150kDa), an d quantum dots (> ~ 20 [Angstrom]), (1,2) and they affect the affinity to bio-molecules. Also, modifications of proteins for attaching tags could vary their biological properties. Furthermore, individual affinity tags require specific buffer conditions, which will affect functions of examined proteins. (3) Therefore, development of low-weight tags, which do not affect the functions of proteins, has been intensively studied. (4,5)
The label-free methods are, therefore, important for studying interactions between native bio-molecules. There are two representative methods. First is the surface plasmon resonance (SPR) biosensor. (6-8) In a conventional SPR measurement, receptor proteins are immobilized on a metallic thin film deposited on a glass plate, which is irradiated from the back surface through a prism by a light, and an analyze is injected. The coupling condition between the evanescent field generated by the incident light and the surface plasmon wave at the metallic film is governed by the macroscopic electric properties in the evanescent field, and the incident angle of the light for exciting SPR changes during the binding reaction between the receptor and target proteins. The incident angle varies as the binding reaction progresses, allowing evaluation of kinetics of the bio-molecule reaction. The SPR method inherently possesses a disadvantage that it is insensitive to reactions, which proceed outside the evanescent field. Therefore, it is difficult to monitor a multi-step interactions or interactions between cells and proteins.
The second method is the quartz-crystal-microbalance (QCM) biosensor. (9-12) Receptor proteins are immobilized on the quartz oscillator surface, and analyte solutions are injected. When target proteins are captured by the receptor proteins, the effective mass of the oscillator increases, resulting in the decrease in the resonance frequency of the oscillator. Then, an injection of washing solution will cause dissociation of the target proteins from receptors, and the resonance frequency will recover. Thus, a real-time monitoring of association and dissociation reactions between bio-molecules is possible, providing their binding affinity. Because the fractional frequency change ([DELTA] f/f) principally equals the ratio of the adsorbed mass to the oscillator mass, (13,14) the QCM biosensor allows quantitative measurement for the binding affinity. Unlike the SPR biosensors, QCM biosensors can detect any mass addition on the quartz surface, and they are applicable to studying interactions between larger biomaterials, such as cells, immobilized on the surface and surrounding proteins.
The sensitivity of the QCM biosensor to the added mass improves as the oscillator mass decreases. Remaining a sufficient active area for biochemical reactions, the reduction of the oscillator mass is achieved by the reduction of its thickness, leading to the increase in the fundamental resonance frequency ([f.sub.1]). Because the amount of the frequency change [absolute value of [DELTA]f] caused by adsorption of proteins is proportional to the square of the fundamental resonance frequency, (11,13) thinning the oscillator significantly increases the sensitivity. Besides, a high-frequency QCM measurement is needed for quantitative analysis. The viscosity effect varies the resonance frequency (15-17) as well as the mass loading effect, making the quantitative evaluation of the adsorbed protein mass complicated. Low-frequency QCM measurements are easily affected by the viscosity effect, and the simultaneous measurement of resonance frequency and dissipation has been performed for compensating for the frequency change due to the viscosity effect. (18-21) Because the viscosity effect becomes insignificant compared with the mass loading effect at high frequencies, (11,15,22) a higher frequency QCM measurement is desired.
Previous studies usually used QCMs with fundamental resonance frequencies lower than 10MHz. (9,10,18-21,23-25) However, higher-frequency QCM systems are recently developed. Natesan et al. (26) used 16.5 MHz QCM for detecting staphylococcal enterotoxin B and achieved the detection limit of 25 ng/mL. Furusawa et al (27,28) developed a 27 MHz QCM system to increase the sensitivity, although their method cannot adopt a flow-injection analysis system. Uttenthaler et al (29) developed high frequency QCMs with resonant frequencies from 39 to 110 MHz in the liquid. Those previous studies needed electrodes on both surfaces of the quartz crystals for applying the effective electric field. However, gold electrodes deteriorate the mass sensitivity of QCM because of gold's mass density much larger than that of quartz, and this influence becomes marked as the fundamental resonance frequency increases. Furthermore, the active sensing region is restricted near the center region where the electrodes are attached, and a large part of surface area remains inactive, deteriorating the sensitivity. Thus, the electrode less QCM biosensors are obviously preferable.
Wireless-electrodeless QCM (WE-QCM) system were recently developed, where a naked ATcut quartz plate was driven by the line antenna or the flat antenna contactlessly, (11) and achieved high frequency QCMs with fundamental resonance frequencies up to 180 MHz. (30) This review article shows the mechanism of the WE-QCM, its usefulness for bioassays, and its extension to micro-electromechanical-system (MEMS) based chip fabrication.
Principle of WE-QCM
Mechanism of QCM biosensor. Figure 1 shows a schematic of the typical use of a QCM biosensor for monitoring the binding reaction between molecules A and B with the association-velocity and dissociation-velocity constants [[kappa].sub.a] and [[kappa].sub.d], respectively. First, the proteins B are immobilized on the quartz-oscillator surface as the receptor proteins, and the fundamental resonance frequency [f.sub.1] is measured in the flowing buffer solution. After the resonance frequency becomes stable, the analyte solution including the molecules A is injected. The molecules A are specifically captured on the oscillator surface by the molecules B, increasing the effective mass of the oscillator and decreasing the resonance frequency. The frequency decreases exponentially when the binding reaction proceeds with a pseudo-first-order manner, (10,14) and the exponential coefficient [alpha] is related to the reaction velocity constants and the concentration of molecule A [C.sub.A] as
[alpha] = [[kappa.sub.a] [C.sub.A] + [[kappa].sub.d] 
Measurements of the exponential coefficients with various analyte concentrations, therefore, allow us to determine the thermodynamic constants, and then the binding affinity (equilibrium constant) [K.sub.A] = [[kappa].sub.a]/[[kappa].sub.d], as well as the concentration of the target protein. Thus, the QCM biosensors enable the label-free assay.
Advantages of WE-QCM. The frequency change [DELTA] [f.sub.1] of the fundamental mode of the quartz oscillator is principally related with the protein mass [[DELTA].sub.m] adsorbed on the quartz surface by the Sauerbrey equation:
[absolute value of [DELTA] [f.sub.1]] = [DELTA]m/[m.sub.q] [f.sub.1]] = [[rho].sub.s][u.sub.q]/2[[rho].sub.q] x 1/[d.sup.2.sub.q]] 
Hence, [m.sub.q], [[rho].sub.q], [v.sub.q], and [d.sub.q] denote the mass, the mass density, the sound velocity (usually of the shear wave), and the thickness of the quartz resonator, respectively. [[rho].sub.s] denotes the area mass density, which is the mass of the adsorbed protein on the unit surface area. Equation  obviously shows that the mass sensitivity ([absolute value of [DELTA][f.sub.1]/[[rho].sub.s]]) of a QCM is inversely proportional to the square of the resonator thickness. (Note that the sensitivity is expressed by [absolute value of [DELTA][f.sub.1]/[[rho].sub.s]] in this paper and this does not equal to the signal-to-noise (S/N) ratio; the S/N ratio highly depends on measurement conditions and should be discussed in individual measurements.) Therefore, thinning the oscillator significantly improves the sensitivity of QCM. However, this has never been straightforward because of the electrodes. Metallic electrodes were indispensable for excitation and detection of vibrations of piezoelectric oscillators. The demand for decreasing the electric resistance has made the electrodes as thick as 100 nm or more. Attachment of such heavy electrodes, however, increases inertia resistance and also mechanical loss due to high damping of metals, deteriorating the sensitivity. Especially, bio-sensing applications often use noble-metal electrodes because they show high affinity to the thiol-mediated self-assembled mono-layers, on which receptor proteins can be immobilized. Mass densities of noble metals (Au: 19,300 kg/[m.sup.3], Pt: 21,400 kg/[m.sup.3]) are much higher than that of quartz (2,648 kg/[m.sup.3]), deteriorating the mass sensitivity, significantly. The influence of the metallic electrodes enlarges at high frequencies, limiting the available frequency with QCM measurements. Figure 2 shows the influences of the gold electrode on quartz surfaces on the frequency change when 1-nm thick human immunoglobulin G (hIgG) layers are adsorbed on both surfaces, displaying the deterioration of the mass sensitivity caused by the electrode. (The details of the calculation appear in Ref. 22.) For example, a 200-nm thick gold electrode lowers the amount of the frequency changes for 170 and 55 MHz QCMs by 45% and 18%, respectively, while it hardly affects the sensitivity of a 5 MHz QCM. Because of a much larger mass density of gold than quartz and the maximum accelerated velocity at the surface, the inertia resistance caused by the electrode becomes significant, making the Sauerbrey equation invalid for a high-frequency QCM with the electrode. Thus, the electrode-less approach is essential not only for a highly sensitive QCM but also for a quantitative analysis with the Sauerbrey equation.
The actual frequency change is caused not only by the mass loading effect, but also by the viscosity effect of the solution (15) and the visco-elastic effect of the protein layer. (16,17) These effects are analyzed using the Voight model (17) for a three-layer model as shown in Fig. 3, consisting of the quartz-oscillator layer, the viscoelastic protein layer, and the viscous solution layer. The results are shown in Fig. 4 for three QCMs with fundamental resonance frequencies of 5, 55, and 180 MHz. The resonance frequencies decrease as the protein adsorption proceeds, and the amount of the frequency change markedly increases for higher frequency QCMs. The frequency change caused by the mass loading is comparable with that caused by the solution viscosity for the low frequency QCM (see upper figures in Fig. 4). Thus, the change in the viscosity of the analyte solution will cause a significant change in the baseline, deteriorating the accuracy of the assay. For the higher-frequency QCMs, the effect is insignificant compared with the mass-loading effect. The visco-elastic properties of the protein layer causes a discrepancy from the Sauerbrey equation for higher frequency QCMs because a very soft layer cannot move on with a high frequency vibration of the substrate (see the blue lines in lower figures in Fig. 4). However, this effect is negligible for standard protein layers composed of high binding bonds with antigen-antibody reaction.
Besides high sensitivity and high quantitative capability, there is a very important advantage in the WE-QCM, that is the replacement-free nature. Other sensor chips used in existing biosensor systems need to be replaced because they use metallic films on their surfaces. The same sensor cannot be used for many times because washing with strong acid or strong alkali deteriorates the adhesion, and metallic films fall off eventually. Biosensor chips must have been replaced, consuming cost and time. Therefore, a replacement-free biosensor has been desired. Because a WE-QCM uses a naked quartz crystal, which is inherently free from the deterioration of electrodes and tolerant to many washing solutions; the quartz crystals can be used permanently. Furthermore, it is revealed that the naked quartz crystal surface adsorbs many proteins with high affinity (31,(32) as shown in Fig. 5, realizing immobilization of receptor proteins directly on the naked quartz surface without linkers such as self-assembled mono-layers. The affinity to streptavidin is especially higher, and using streptavidin base on quartz, we can detect any analyte via biotin-conjugated receptors on streptavidin, where the sensor chip never needs to be replaced as shown in Fig. 6. Because inactive proteins such as bovine serum albumin (BSA) also adsorbs on the naked quartz surfaces, the uncovered surface regions can be blocked by injecting them after receptor proteins as will be seen in Fig. 14.
Measurement setup. In WE-QCM systems, two line or flat antennas are used for excitation and detection of the shear vibrations of the AT-cut quartz oscillator. Figure 7 shows an example of the sensor cell for a multi-channel WE-QCM. The AT-cut quartz oscillators with different thicknesses are aligned along the flow channel. They are lightly sandwiched by two silicon-rubber gaskets. The two line antennas are located outside the flow channel. The tone bursts are applied to the generation antenna, which causes the shear vibrations of the oscillators through the electromagnetic field. After the excitation, the vibrations are detected by the detection antenna through the electromagnetic fields induced by the vibrations. Thus, the non-contacting measurement of the oscillators' vibrations is achieved with this measurement setup.
Figure 8 shows the electronics to measure the resonance frequency contactlessly by the antennas. A superheterodyne spectroscopy (33,34) was developed with the intermediate frequency ([[omega].sub.TF]) of 25 MHz, which is provided by the local synthesizer. The master synthesizer generates a sinusoidal signal with the frequency of [[omega].sub.IF] + [[omega].sub.D], where [[omega].sub.D] is the driving frequency and it is set to be close to the resonance frequency of the quartz oscillator. The outputs from the two synthesizers enter the first multiplier and the driving-frequency signal with the frequency [[omega].sub.D] is produced by the following low-pass filter. After the high-power gated amplifier, the bust signal is fed to the antenna wire for generation, which induces the electric field to cause pure-shear oscillation in the quartz crystal via the converse piezoelectric effect. (35) After the excitation, the antenna wire for detection receives the reverberating signal of the oscillator vibration with the resonance frequency [[omega].sub.R]. This signal and the reference signal from the master synthesizer enter the second multiplier and the output signal is fed to the third multiplier after the bandpass filter set at [[omega].sub.IF]. The reference signal from the local synthesizer also enters the third multiplier, and the output signal of the differential frequency component [DELTA][omega] = [[omega].sub.R] - [[omega].sub.R] is extracted by the low-pass filter. This beating signal is integrated by the analog integrator, and the final output is stored in the personal computer to calculate the resonance frequency of the quartz oscillator [[omega].sub.R]. Figure 9 shows an example of measured resonance spectrum for four channel WE-QCM.
The sensor cell is installed in a homebuilt flow-injection-analysis system. An analyte solution is selected and injected by a switching valve among several, which replaces the carrier buffer solution. A steady flow with the flow rate between 100 and 500 [micro]l/min is supplied by a micro-pump. A degasifier is located before the micro-pump to decrease the solvent gases in the solution. After the micro-pump, the solution flows in a 3m Teflon tube column, which maintains the temperature of the solution at 37[degrees]C. In the sensor cell, the solution flows along both surfaces of the sensor chips. Note that a WE-QCM can use both surfaces as the sensing region.
High sensitivity of WE-QCM
Figure 10 shows typical example for demonstrating the high sensitivity and wide dynamic range of the WE-QCM. (22) Staphylococcal protein A (SPA) was immobilized on the both surfaces of quartz resonators, and hIgG solutions with various concentrations between 5 pM and 50 nM were injected. Obviously, the sensitivity of the 170 MHz QCM biosensor is much higher than that of a conventional 5 MHz QCM biosensors. According to the Sauerbrey equation, the mass sensitivity of a QCM considering the electrode area [A.sub.e] is given by
[absolute value of [DELTA][f.sub.1]/[[rho].sub.s]]] = [A.sub.e][u.sub.q]/2[A.sub.q][[rho].sub.q] x [1/ [d.sup.2.sub.q]] 
where [A.sub.q] denotes the one-sided surface area of the quartz crystal. Thus, the ratio [A.sub.e]/[A.sub.q] is directly related to the QCM sensitivity, and it is smaller than 1 for a conventional QCM using a single side of a quartz plate, while it nearly equals 2 for the wireless-electrodeless QCM because almost whole both surfaces can be used. Figure 11 shows a relationship between the fundamental resonance frequency and the mass sensitivity ([absolute value of [DELTA][f.sub.1]/[[rho].sub.s]]) for conventional QCMs and WE-QCMs. The mass sensitivities are 0.056, 0.18, and 1.64Hz x [cm.sup.2]/ng for 5, 9, and 27 MHz conventional QCMs, respectively, whereas they are 6.8, 14, 65, and 73Hz x [cm.sup.2]/ng for 55, 80, 170, and 180 MHz WE-QCMs, respectively. Thus, the mass sensitivity for a 170 MHz electrode-less QCM is three-orders-of-magnitude larger than that of a conventional 5-MHz single-side QCM. With the 170 MHz WE-QCM, the label-free direct detection of hIgG with a concentration of 0.5 pM was possible, which will be only achieved with this technique. The measured [absolute value of [DELTA][f.sub.1]] for the injection of 50 nM hIgG solution were 62, 7450, and 61000 Hz for 5, 55, and 170 MHz WE-QCMs, respectively, yielding the linear relationship between [f.sub.1.sup.2] and [DELTA][f.sub.1] with the coefficient of correlation of [absolute value of R] = 0.9999. (22) This agreement with the Sauerbrey equation confirms quantitative aspect of the WE-QCM.
The high sensitivity of a WE-QCM allows evaluation of concentration dependence of binding affinity between bio-molecules. (36) Figure 12, for example, plots the relationship between the inversed frequency change [DELTA][f.sub.e.sup.-1] at the equilibrium state and the inversed concentration of the injected analyte (IgG) [C.sub.IgG.sup.-1]. The reaction kinetics theory predicts a linear relationship between them, and the equilibrium constant [K.sub.A] is given from its slope and intercept. (10) A single line, however, failed to fit to the results for a wide concentration range (the broken line), indicating the dependence of Ka on the concentration of analyte. The [K.sub.A] value takes 6.2 x [10.sup.6], 9.0 x [10.sup.8], and 6.0 x [10.sup.10] [M.sup.-1] for concentration ranges between 5 and 20 [micro]g/ml, 0.1 and 5 [micro]g/ml, and 1 and 100ng/ml, respectively. The affinity value thus significantly varies depending on the analyte concentration. The concentration dependence of the affinity between IgG and protein A was attributed to the different structures of the IgG-protein A complex. Hanson and Schumaker (37) suggested five plausible structures for IgG-protein A complexes, depending on their molar ratio. Individual complex structures should possess different affinity.
The high-sensitive WE-QCM contributes to avoid deterioration of the apparent affinity due to the hindrance effect. Figure 13 shows binding curves for injections of hIgG solutions of various concentrations on the SPA-immobilized sensor chip. (38) Solid lines are the fitted theoretical function. When an hIgG solution arrives at the sensor cell, the frequency starts to decrease exponentially. The theoretical exponential function with a single exponential coefficient given by Eq.  well fitted the measurements for lower-concentration solutions. For higher-concentration solutions (> 34 nM), however, a single-exponential function failed to fit the observed frequency change, and two exponential functions were apparently required to explain the frequency change. The critical value, at which the frequency change departs from the first exponential function, appeared around [DELTA] f/f = 9.5 x [10.sup.-5] ([DELTA]f = 5.2kHz).
This critical value was consistently explained from the steric hindrance effect of hIgG molecules. At the critical point, almost the whole surfaces of the oscillator were covered by hIgG molecules, and the steric hindrance sub-sequently occurred, resulting in deceleration of binding affinity because of buried binding sites of protein A. This result gave an important indication in determining the kinetics constants using a flow injection system. Previous QCMs used only one side of their oscillators, and when a conventional 9 MHz QCM is used, for example, the critical value will be [DELTA]f/f = -7.8 x [10.sup.-6] or [DELTA] f = -70 Hz. Therefore, the affinity evaluation must be performed using small frequency changes; otherwise, the affinity value will be underestimated due to the steric hindrance effect. Furthermore, not only QCMs but also SPR immunoassays adopted flow injection systems to evaluate the kinetics of bio-molecule reactions, and they have to use low-concentration analyte solutions before the steric hindrance effect becomes dominant. Therefore, using a high-sensitive QCM is the key for accurately determining the binding affinity between bio-molecules.
One of the central issues in the QCM study is development of the multi-channel QCM (MQCM) biosensor. It increases the efficiency of detection of target proteins and enables monitoring of interactions among three or more bio-molecules. Intensive efforts have been, therefore, paid for establishing MQCM biosensors. This MQCM is capable of monitoring the biological reactions in the same liquid environment. Using one channel as a reference, it allows compensation for temperature and viscosity effects. However, the ineluctable interference among channels has been an unsolved problem. The principal approach was developing the one-chip MQCM, where several sensing regions were prepared on a single quartz plate. Tatsuma et al (39) made four sensor regions by chemically etching the quartz plate and confirmed their independent operation by dropping a water droplet. Abe et al. (40-42) fabricated different-thickness sensing regions in a single quartz plate by the deep-reactive ion etching method. They demonstrated a four-channel measurement as a gas sensor. Jin et al. (43) also developed a four-channel one-chip MQCM and adopted it to a gas sensor. However, because of the ineluctable interference among channels, the development of an MQCM for an immunosensor has been difficult with this approach. Another approach was the combination of many single-channel QCMs, (44) where the interference is negligible. However, it required a larger amount of solutions, and the liquid environment (temperature, pressure, and so on) will not be the same among sensor cells, downplaying the important advantage of the MQCM. Also, it is not easy to increase the number of the channel, because many instruments were needed for individual channels.
A wireless-electrode-less multi-channel QCM (WE-MQCM) was then developed to solve the difficulties. (45) The shear vibrations of AT-cut quartz-crystal plates with different thicknesses were simultaneously excited and detected by the same antenna in the same reaction cell. Because of the independent sensor chips, the frequency interference among chips is negligible, while the reactions can be monitored in the same environment. The electrode-less blank quartz oscillators were set in a line along the flow channel. The thicknesses of the quartz plates were varied between 39 and 30 [micro]m, corresponding to 43 and 55 MHz fundamental resonance frequencies, respectively. Up to a 10-channel WE-MQCM was developed, and further increase of the channel number is easily achieved using smaller crystals. The nonspecific binding was used for immobilizing the receptor proteins, which remained the replacement-free nature.
Figure 14 shows examples of application of a five-channel WE-MQCM for detecting different antigen-antibody reactions. Anti hIgG antibody (AhIgG), anti mouse IgG antibody (AmIgG), anti rabbit IgG antibody (ArIgG), SPA, and BSA were immobilized nonspecifically as shown in Fig. 14, and 50 nM analyte solutions were injected. Figures 14(a)-(c) show the frequency changes observed in the five-channel WE-MQCM measurements responding to injections of hIgG, mIgG, and rIgG solutions, respectively, where the frequency change in the reference channel was subtracted. The channel with the antibody receptor showed a significantly larger frequency decrease when the corresponding antigen was injected, while other channels showed lower frequency changes except for the SPA channel. The SPA binds with IgG molecules specifically, and the number of IgG molecule which can bind with a single SPA is larger than one. (37) Thus, the amount of the frequency change in the SPA channel was always larger than the other channels. rIgG especially shows higher affinity with SPA, (46) resulting in the largest frequency decrease. The simultaneous measurement allowed to observe nonspecific adsorptions. For example, AhIgG binded with rIgG with relatively higher affinity. Thus, the WE-MQCM is useful for studying interactions among proteins. A further increase of the channel number will require reducing the size of the quartz plate for setting all the crystals in the same environment.
Aggregation reaction of amyloid [beta] peptides
Aggregation processes of amyloid [beta] (A [beta]) peptides are deeply involved in pathogenic mechanisms of Alzheimer's disease (AD). (47-49) There are two principal full-length AO peptides related to AD, A [[beta].sub.1-40] (A [beta] 40) and A [beta].sub.1-42] (A [beta] 42), which are extracted from the parental amyloid precursor proteins by enzymes or enzyme complexes. (50) A [beta] 40 is predominantly produced, but A [beta] 42 is more hydrophobic to cause fibril formation (51) and more neurotoxic. (52) Thus, understanding of the aggregation mechanism of the A[beta] peptides remains a central issue in the peptide polymerization study. Previous works studied their aggregation process in bulk solutions, (51,53-56) where interactions among nuclei, aggregates, and monomers dominate the polymerization process for oligomers and fibrils in this case, blinding the most important interaction between nuclei and surrounding peptides. Thus, it is important to systematically study deposition behaviors of peptides on nuclei without involving interactions between nuclei.
A WE-MQCM was then applied to systematically study the deposition behaviors of A[beta]40 and A[beta]42 monomers on various nuclei over 40 h. (57,58) Figure 15 shows a schematic of such measurements. A[beta]40 and A[beta]42 nuclei grown at pH 7.4 or 4.6 were immobilized on the quartz surfaces, and the monomer A[beta] solution was injected. Figure 16 shows examples of homogeneous-deposition behaviors on the nuclei grown at pH 7.4, displaying successful monitoring of the deposition reactions for a long time. The most important observation was that A[beta]40 peptides markedly accumulate on the A[beta]42 nuclei grown at pH 4.6 (Fig. 17), resulting in oligomeric deposits (Fig. 18). This deposition rate is much higher than those in other cases (Fig. 17(b). From these results, an important model for AD was proposed as follows. (57) Oli gomers and related structures are more neurotoxic than fibrils, (59-61) and significant aggregation behavior for oligomeric structures is the key of the AD mechanism. A[beta]42 is more hydrophobic and will self-associate just after its production from the amyloid precursor protein by the cleaving enzymes ([beta] and [gamma] secretases (50), which show high activity at lower pH. Therefore, the isolated A[beta]42 peptides can form the active nuclei immediately at a lower pH environment, and they diffuse inside and outside cells for neutral pH environments, with which A[beta]40 peptides interact more frequently, because most of the produced peptides is A[beta]40 (~90%). (62)-(64) Thus, the large amount of A[beta]40 peptides deposit on the A[beta]42 nuclei to cause and grow oligomeric structures.
Frequency responses in Figs. 16 and 17 are not so smooth as those observed for other binding systems. Unlike a usual antigen-antibody binding reaction with a single equilibrium constant, accumulation reaction of A[beta] peptides progresses with much more complexity. For example, multiple kinetics will appear including the fibril-end kinetics for transition into the active end and the kinetics for monomer attachment. In this case, an uneven frequency response is expected even in the macroscopic measurement, because all monomers are simultaneously dissolved to grow nuclei. Similar multi-kinetics mechanism will appear in the case of oligomer formation. Thus, the non-smooth frequency change will be a characteristic in the deposition reaction of the A[beta] peptides. (58)
While a thinner quartz oscillator achieves a high-sensitive QCM biosensor, it is fragile and easy to break up during its installation in the sensor cell. Also, in a WE-QCM setup, a part of the quartz plate was fixed by sandwiching with two silicon-rubber gaskets, deteriorating the Q value (~800 at 170 MHz at most). As a breakthrough to these problems, the resonance acoustic microbalance with naked embedded quartz (RAMNE-Q) chip was proposed. (65-67) It embeds a bare thin quartz oscillator in a micro-channel fabricated in Si wafer by the micro-electromechanical system (MEMS) technology, achieving a reusable high-frequency (fundamental resonance frequency of 178 MHz) and high-Q (~1500) biosensor.
A RAMNE-Q chip is composed of three substrates (glass/silicon/glass) as shown in Figs. 19(a) and (b). The Si micro-channel is fabricated by inductively coupled plasma reactive-ion etching (ICP-RIE) into the middle silicon substrate, and a naked AT-cut quartz oscillator is embedded in the micro-channel, which is supported by the micro-pillars and micro-walls without fixed parts as illustrated in Figs. 19(c) and (d). The quartz oscillator becomes stable with the point-contact supports even in a solution-flow state. The glass substrates are bonded to both sides of the silicon substrate by the anodic-bonding method. This biosensor chip remains reusable owing to the integration with a rigid package; a washing procedure releases the bonded proteins from the surfaces, making the naked surfaces repeatedly. The structural damping is restricted by the minimized contacts (point contacts) with the quartz oscillator, achieving a high-Q value (~1500) in the solution flow. Figure 20 shows the appearance of the fabricated RAMNE-Q biosensors before the singulation from the ware.
Figure 21 displays the usability of a RAMNE-Q biosensor. A real-time monitoring for detecting hIgG molecules was performed using nonspecifically immobilized SPA molecules on the quartz oscillator. The hIgG concentration was between 0.001-10 [micro]g/ml. A glycin-HCl buffer solution was used for dissociating hIgG from SPA after each binding reaction. The maximum frequency change is shown by the dashed line in Fig. 21(a) for the injection of 10ng/ml hIgG solution when the SPA molecule was absent, which corresponds to the negative (nonspecific) binding assay. Thus, the specific biding causes significantly larger frequency change (~20kHz for 10ng/ml) than the nonspecific binding (0.05 kHz).
It is appropriate to use Eq.  for evaluating the concentration because it is independent of the amount of the receptor protein on the surface. Figure 21(b) shows the a values obtained for different-concentration hIgG solutions, showing a good correlation with the hIgG concentration. The same RAMNE-Q chip was used for many measurements; it was washed before the measurement every time. Nevertheless, identical results were obtained as shown with the error bars in Fig. 21(b), showing the high reproducibility of a single RAMNE-Q biosensor.
Sensitivity amplification with sandwich assays
As an important practical use, QCM biosensors are expected to detect biomarkers. However, human serum contains many blood proteins, and they are adsorbed on the sensor chip surfaces nonspecifically, making it difficult to extract from the total frequency response the small frequency change caused by the binding of a target protein with the receptor on the surface. Also, threshold concentrations are usually lower than the detection limit of QCM biosensors. To overcome these difficulty, mass-amplified sandwich-assay methods were proposed. Gold nanoparticles were, for example, used for the mass amplification in QCM assays. (68-70) However, high affinity of gold for proteins (32) causes nonspecific adsorption on receptor proteins. Also, preparation of surface-modified particles takes longer. In the WE-QCM, a mass-amplification method using streptavidin was proposed, (30) where streptavidin molecules quickly combines with biotins with extremely high affinity. As a target protein, the C-reactive protein (CRP) was adopted, which is an important biomarker for checking inflammation. Its threshold level is about 30 ng/ ml, and a very quick assay is often required in an emergency case to judge the cause of illness.
Figure 22 shows the schematic of the sandwich assay with the mass-amplified detection antibody. Because a single streptavidin has four binding sites with biotin, it can connect more than two antibody molecules through biotin terminals. The molecular mass of the detection-antibody complex was estimated to be 3.2 MDa, corresponding to a complex of six detection antibodies with seven streptavidins attached to each antibody. Figure 23 shows an example of the enhanced frequency change by the mass-amplified detection-antibody solution. Injection of the 1 ng/ ml CRP solution caused only a 200-Hz frequency decrease, and the baseline increased after 15 min from the injection. Thus, it would have been difficult to estimate the amount of the frequency change caused by the binding of CRP with the capturing antibody. The injection of the mass-amplified detection-antibody solution, however, caused nearly a 5-kHz frequency change, demonstrating the successful amplification of the mass sensitivity. Figure 24 shows the binding curves observed after the arrival of the mass-amplified detection-antibody solution, which was followed by injection of CRP solutions and the PBS solution. The frequency decrease was observed even for the injection of the detection-antibody solution after the analyte solution without CRP molecules (0ng/ml), indicating the nonspecific binding of the detection-antibody complex onto the capturing antibody. However, this frequency change was significantly smaller than those observed for the analyte solutions containing CRP molecules. The inset in Fig. 24 shows the correlation between the frequency decrement at 20 min after the arrival time of the mass-amplified detection-antibody solution and the concentration of the CRP solution. Such a correlation was obtained when antibodies from the same production lots were used; the frequency change fluctuated among assays from different production lots. (30)
The wireless-electrode-less quartz-crystal-microbalance biosensors have shown great capabilities of quantitatively studying interactions between bio-molecules with high sensitivity (large frequency change). Its replacement-free nature will allow us to install it into civilian goods for health and food monitoring. Its sensitivity will be further improved with the RAMNE-Q technology, which also yields high reproducibility and the price reduction with the mass production. The electrode-less aspect of the WE-QCM easily allows its integration with a high-resolution optical methods such as the total internal reflection fluorescence microscopy and confocal microscopy. Therefore, the WE-QCM will be a key measurement tool in life-science studies and diagnosis in near future.
The author is deeply grateful to Professor Masahiko Hirao at Osaka University for his continuous help for this research and to Dr. Fumihito Kato at Osaka University for his help for fabricating RAMNE-Q biosensors.
(Received July 2, 2013; accepted Sep. 2, 2013)
(1) Green, M. (2004) Semiconductor quantum dots as biological imaging agents. Angew. Chem. Int. Ed. Engl. 43, 4129-4131.
(2) Bruchez, M., Moronne, M., Gin, P., Weiss, S. and Alivisatos, A. (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013-2016.
(3) Terpe, K. (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533.
(4) Guignet, E., Hovius, R. and Vogel, H. (2004) Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22, 440-444.
(5) Ojida, A., Honda, K., Shinmi, D., Kiyonaka, S., Mori, Y. and Hamachi, I. (2006) Oligo-asp tag/ Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins. J. Am. Chem. Soc. 128, 10452-10459.
(6) Nedelkov, D. (2007) Development of surface plasmon resonance mass spectrometry array platform. Anal. Chem. 79, 5987-5990.
(7) Hoa, X.D., Kirk, A.G. and Tabrizian, M. (2007) Towards integrated and sensitive surface plasmon resonance biosensors: A review of recent progress. Biosens. Bioelectron. 23, 151-160.
(8) Kurita, R., Yokota, Y., Ueda, A. and Niwa, O. (2007) Electrochemical surface plasmon resonance measurement in a micro-liter volume flow cell for evaluating the affinity and catalytic activity of bio-molecules. Anal. Chem. 79, 9572-9576.
(9) Muramatsu, H., Dicks, M., Tamiya, E. and Karube, I. (1987) Piezoelectric crystal biosensor modified with protein A for determination of immuno globulins. Anal. Chem. 59, 2760-2763.
(10) Liu, Y., Yu, X., Zhao, R., Shangguan, D., Bo, Z. and Liu, G. (2003) Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents. Biosens. Bioelectron. 19,9-19.
(11) Ogi, H., Motohisa, K., Matsumoto, T., Hatanaka, K. and Hirao, M. (2006) Isolated electrodeless high frequency quartz crystal microbalance for immunosensors. Anal. Chem. 78, 6903-6909.
(12) Pei, Y., Yu, H., Pei, Z., Theurer, M., Ammer, C., Andre, S., Gabius, H.-J., Yan, M. and Ramstrom, O. (2007) Photo-derivatized polymer thin films at quartz crystal microbalance surfaces: sensors for carbohydrate-protein interactions. Anal. Chem. 79, 6897-6902.
(13) Sauerbrey, G. (1959) Verwendung von Schwing quarzen zur Wagung diinner Schichten und zur Mikrowagung. Z. Phys. 155, 206-222.
(14) Eddowes, M.J. (1987) Direct immunochemical sensing: basic chemical principles and fundamental limitations. Biosensors 3,1-15.
(15) Kanazawa, K. and Gordon, J. (1985) Frequency of a quartz microbalance in contact with liquid. Anal. Chem. 57, 1770-1771.
(16) Reed, C.E., Kanazawa, K.K. and Kaufman, J.H. (1993) Physical description of a visco-elastically loaded AT-cut quartz resonator. J. Appl. Phys. 68,1993-2001.
(17) Voinova, M.V., Jonson, M. and Kasemo, B. (1997) Dynamics of viscous amphiphilic films supported by elastic solid substrates. J. Phys. Condens. Matter 9, 7799-7808.
(18) Hook, F., Kasemo, B., Nylander, T., Fant, C., Sott, K. and Elwing, H. (2001) Variations in coupled water, viscoelastic properties, and film thickness of a mefp-1 protein film during adsorption and cross linking: A quartz crystal microbalance with dissipation monitoring, ellipsometry, and surface plasmon resonance study. Anal. Chem. 73, 5796-5804.
(19) Jonsson, M.P., Jonsson, P. and Hook, F. (2008) Simultaneous nanoplasmonic and quartz crystal microbalance sensing: Analysis of bio-molecular conformational changes and quantification of the bound molecular mass. Anal. Chem. 80, 7988-7995.
(20) Patel, A.R., Kanazawa, K.K. and Frank, C.W. (2009) Antibody binding to a tethered vesicle assembly using QCM-D. Anal. Chem. 81, 6021-6029.
(21) Yan, M., Liu, C., Wang, D., Ni, J. and Cheng, J. (2011) Characterization of adsorption of humic acid onto alumina using quartz crystal microbalance with dissipation. Langmuir 27, 9860-9865.
(22) Ogi, H., Nagai, H., Fukunishi, Y., Hirao, M. and Nishiyama, M. (2009) 170-MHz electrodeless quartz crystal microbalance biosensor: Capability and limitation of higher frequency measurement. Anal. Chem. 81, 8068-8073.
(23) Guzmn, E., Ritacco, H., Ortega, F., Svitova, T., Radke, C.J. and Rubio, R.G. (2009) Adsorption kinetics and mechanical properties of ultra-thin polyelectrolyte multi-layers: Liquid-supported versus solid-supported films. J. Phys. Chem. B 113, 7128-7137.
(24) Kang, H. and Muramatsu, H. (2009) Monitoring of cultured cell activity by the quartz crystal and the micro CCD camera under chemical stressors. Biosens. Bio-electron. 24, 1318-1323.
(25) Wang, S., Milam, J., Ohlin, A.C., Rambaran, V.H., Clark, E., Ward, W., Seymour, L., Casey, W.H., Holder, A.A. and Miao, W. (2009) Electrochemical and electrogenerated chemiluminescent studies of a trinuclear complex, [([(phen).sub.2]Ru[(dpp)).sub.2]Rh[Cl.sub.2][5.sup.+], and its interactions with calf thymus DNA. Anal. Chem. 81, 4068-4075.
(26) Natesan, M., Cooper, M.A., Tran, J.P., Rivera, V.R. and Poli, M.A. (2009) Quantitative detection of staphylococcal enterotoxin B by resonant acoustic profiling. Anal. Chem. 81, 3896-3902.
(27) Furusawa, H., Komatsu, M. and Okahata, Y. (2009) In situ monitoring of conformational changes of and peptide bindings to calmodulin on a 27 MHz quartz-crystal microbalance. Anal. Chem. 81, 1841-1847.
(28) Furusawa, H., Ozeki, T., Morita, M. and Okahata, Y. (2009) Added mass effect on immobilizations of proteins on a 27 MHz quartz crystal microbalance in aqueous solution. Anal. Chem. 81, 2268-2273.
(29) Uttenthaler, E., Schr'aml, M., Mandel, J. and Drost, S. (2001) Ultrasensitive quartz crystal microbalance sensors for detection of M13-Phages in liquids. Biosens. Bioelectron. 16, 735-743.
(30) Ogi, H., Yanagida, T., Hirao, M. and Nishiyama, M. (2011) Replacement-free mass-amplified sandwich assay with 180-MHz electrodeless quartz-crystal microbalance biosensor. Biosens. Bioelectron. 26, 4819-4822.
(31) Ogi, H., Okamoto, K., Nagai, H., Fukunishi, Y. and Hirao, M. (2009) Replacement-free electrodeless quartz crystal microbalance biosensor using nonspecific-adsorption of streptavidin on quartz. Anal. Chem. 81, 4015-4020.
(32) Ogi, H., Fukunishi, Y., Nagai, H., Okamoto, K., Hirao, M. and Nishiyama, M. (2009) Non-specific adsorption behavior of polyethylenglycol and bovine serum albumin studied by 55-MHz wireless-electrode less quartz crystal microbalance. Biosens. Bio-electron. 24, 3148-3152.
(33) Hirao, M., Ogi, H. and Fukuoka, H. (1993) Reso nance EMAT system for acoustoelastic stress evaluation in sheet metals. Rev. Sci. Instrum. 64, 3198-3205.
(34) Petersen, G.L., Chick, B.B., Fortunko, C.M. and Mirao, M. (1994) Resonance techniques and apparatus for elastic-wave velocity determination in thin metal plates. Rev. Sci. Instrum. 65, 192-198.
(35) Ogi, H., Niho, H. and Hirao, M. (2006) Internal friction mapping on solids by resonance ultrasound microscopy. Appl. Phys. Lett. 88, 141110.
(36) Ogi, H., Motohisa, K., Hatanaka, K., Ohmori, T., Hirao, M. and Nishiyama, M. (2007) Concentration dependence of IgG-protein A affinity studied by wireless-electrodeless QCM. Biosens. Bioelectron. 22, 3238-3242.
(37) Hanson, D.C. and Schumaker, V.N. (1984) A model for the formation and inter-conversion of protein A-immunoglobulin G soluble complexes. J. Immunol. 132, 1397-1409.
(38) Ogi, H., Fukunishi, Y., Omori, T., Hatanaka, K., Hirao, M. and Nishiyama, M. (2008) Effects of flow rate on sensitivity and affinity in flow injection biosensor systems studied by 55-MHz wireless quartz crystal microbalance. Anal. Chem. 80, 5494-5500.
(39) Tatsuma, T., Watanabe, Y. and Oyama, N. (1999) Multi-channel quartz crystal microbalance. Anal. Chem. 71, 3632-3636.
(40) Abe, T. and Esashi, M. (2000) One-chip multi channel quartz crystal microbalance (QCM) fabricated by Deep RIE. Sens. Actuators A Phys. 82,139-143.
(41) Hung, V.N., Abe, T., Minh, P.N. and Esashi, M. (2002) Miniaturized, highly sensitive single-chip multi-channel quartz-crystal microbalance. Appl. Phys. Lett. 81, 5069-5071.
(42) Hung, V.N., Abe, T., Minh, P.N. and Esashi, M. (2003) High-frequency one-chip multi-channel quartz crystal microbalance fabricated by deep RIE. Sens. Actuators A Phys. 108,91-96.
(43) Jin, X., Huang, Y., Mason, A. and Zeng, X. (2009) Multi-channel monolithic quartz crystal microbalance gas sensor array. Anal. Chem. 81 , 595-603.
(44) Zhang, B., Mao, Q., Zhang, X., Jiang, T., Chen, M., Yu, F. and Fu, W. (2004) A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin. Biosens. Bio-electron. 19, 711-720.
(45) Ogi, H., Nagai, H., Fukunishi, Y., Yanagida, T., Hirao, M. and Nishiyama, M. (2010) Multi-channel wireless-electrodeless quartz-crystal microbalance immunosensor. Anal. Chem. 82, 3957-3962.
(46) Hanson, D.C., Phillips, M.L. and Schumaker, V.N. (1984) Electron microscopic and hydrodynamic studies of protein A-immuno globulin G soluble complexes. J. Immunol. 132, 1386-1396.
(47) Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos, M., Morgan, T.E., Rozovsky, I., Trommer, B., Viola, K.L., Wals, P., Zhang, C., Finch, C.E., Krafft, G.A. and Klein, W.L. (1998) Diffusible, non-fibrillar ligands derived from AO1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. U.S.A. 95, 6448-6453.
(48) Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M. and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507-511.
(49) Stefani, M. and Dobson, C.M. (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. 81, 678-699.
(50) LaFerla, F.M., Green, K.N. and Oddo, S. (2007) Intracellular amyloid-beta in Alzheimer's disease. Nat. Rev. Neurosci. 8, 499-509.
(51) Jarrett, J.T., Berger, E.P. and Lansbury, P.T. Jr. (1993) The carboxy terminus of the O amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease. Biochemistry 32, 4693-4697.
(52) Takahashi, R.H., Milner, T.A., Li, F., Nam, E.E., Edgar, M.A., Yamaguchi, H., Beal, M.F., Xu, H., Greengard, P. and Gouras, G.K. (2002) Intraneuronal Alzheimer AO42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am. J. Pathol. 161, 1869-1879.
(53) Wood, S.J., Maleeff, B., Hart, T. and Wetzel, R. (1996) Physical, morphological and functional differences between pH 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide AO. J. Mol. Biol. 256, 870-877.
(54) Nybo, M., Svehag, S.-E. and Nielsen, E.H. (1999) An ultrastructural study of amyloid intermediates in A[[beta].sub.1]-42 fibrillogenesis. Scand. J. Immunol. 49, 219-223.
(55) Bitan, G., Kirkitadze, M.D., Lomakin, A., Vollers, S.S., Benedek, G.B. and Teplow, D.B. (2003) Amyloid O-protein (AO) assembly: AO40 and AO42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. U.S.A. 100, 330-335.
(56) Benseny-Cases, N., Cocera, M. and Cladera, J. (2007) Conversion of non-fibrillar [beta]-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation. Biochem. Biophys. Res. Commun. 361, 916-921.
(57) Ogi, H., Fukunishi, Y., Yanagida, T., Yagi, H., Goto, Y., Fukushima, M., Uesugi, K. and Hirao, M. (2011) Seed-dependent deposition behavior of A[beta] peptides studied with wireless quartz-crystal microbalance biosensor. Anal. Chem. 83, 4982-4988.
(58) Ogi, H., Fukushima, M., Uesugi, K., Yagi, H., Goto, Y. and Hirao, M. (2013) Acceleration of deposition of A[[beta].sub.1-40] peptide on ultrasonically formed A[[beta].sub.1-42] nucleus studied by wireless quartz-crystal-microbalance biosensor. Biosens. Bioelectron. 40, 200-205.
(59) Klein, W.L., Krafft, G.A. and Finch, C.E. (2001) Targeting small AO oligomers: the solution to an Alzheimer's disease conundrum? Trends Neurosci. 24, 219-224.
(60) Mastrangelo, I.A., Ahmed, M., Sato, T., Liu, W., Wang, C., Hough, P. and Smith, S.O. (2006) High resolution atomic force microscopy of aoluble AO42 oligomers. J. Mol. Biol. 358, 106-119.
(61) Walsh, D.M., Klyubin, I., Fadeeva, J.V., Cullen, W.K., Anwyl, R., Wolfe, M.S., Rowan, M.J. and Selkoe, D.J. (2002) Naturally secreted oligomers of amyloid protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539.
(62) Suzuki, N., Cheung, T.T., Cai, X.-D., Odaka, A., Otvos, L. Jr., Eckman, C., Golde, T.E. and Younkin, S.G. (1994) An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336-1340.
(63) Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N. and Ihara, Y. (1994) Visualization of A[beta]42(43) and A[beta]40 in senile plaques with end specific A[beta] monoclonals: Evidence that an initially deposited species is A[beta]42(43). Neuron 13,45-53.
(64) Gravina, S.A., Ho, L.B., Eckman, C.B., Long, K.E., Otvos, L. Jr., Younkin, L.H., Suzuki, N. and Younkin, S.G. (1995) Amyloid [beta] protein (A[beta])in Alzheimer's disease brain. J. Biol. Chem. 270, 7013-7016.
(65) Kato, F., Ogi, H., Yanagida, T., Nishikawa, S., Nishiyama, M. and Hirao, M. (2011) High frequency electrodeless quartz crystal microbalance chip with a bare quartz resonator encapsulated in a silicon microchannel. Jpn. J. Appl. Phys. 50, 07HD03.
(66) Kato, F., Ogi, H., Yanagida, T., Nishikawa, S., Hirao, M. and Nishiyama, M. (2012) Resonance acoustic microbalance with naked-embedded quartz (RAMNE-Q) biosensor fabricated by micro-electromechanical-system process. Biosens. Bio-electron. 33, 139-145.
(67) Kato, F., Tsurimoto, K., Ogi, H. and Hirao, M. (2013) Application of sandwich assay to resonance acoustic microbalance with naked-embedded quartz biosensor for high-selectivity detection of C-reactive protein. Jpn. J. Appl. Phys. 52,07HD11.
(68) Mao, X., Yang, L., Su, X.-L. and Li, Y. (2006) A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7. Biosens. Bio-electron. 21, 1178-1185.
(69) Zhu, Z., Su, Y., Li, J., Li, D., Zhang, J., Song, S., Zhao, Y., Li, G. and Fan, C. (2009) Highly sensitive electrochemical sensor for mercury(II) ions by using a mercury-specific oligonucleotide probe and gold nanoparticle-based amplification. Anal. Chem. 81, 7660-7666.
(70) Chen, Q., Tang, W., Wang, D., Wu, X., Li, N. and Liu, F. (2010) Amplified QCM-D biosensor for protein based on aptamer-functionalized gold nanoparticles. Biosens. Bioelectron. 26, 575-579.
By Hirotsugu OGI (*1),([dagger])
(Communicated by Shun-ichi IWASAKI, M.J.A.)
(*1) Graduate School of Engineering, Osaka University, Osaka, Japan.
([dagger]) Correspondence should be addressed: H. Ogi, Graduate School of Engineering, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan (e-mail: email@example.com).
Hirotsugu Ogi was born in 1967 in Kyoto, Japan. He received BS and MS degrees from Osaka University in mechanical engineering in 1991 and 1993, respectively, and became a research associate at Osaka University in 1993. He received his Ph.D. in 1997 from Osaka University for the study on electromagnetic acoustic resonance for materials characterization. He was invited from National Institute of Standards and Technology in U.S.A. as a guest researcher for fourteen months from July in 1997, where he studied on elastic and anelastic properties of materials. He became an Associate Professor at Osaka University in 2000. He currently studies materials science and bioscience with his originally developed acoustic methods. He was awarded JSPS Prize in 2012 for study on high-sensitive acoustic biosensors.
|Printer friendly Cite/link Email Feedback|
|Publication:||Japan Academy Proceedings Series B: Physical and Biological Sciences|
|Date:||Sep 1, 2013|
|Previous Article:||On earthquake prediction in Japan.|
|Next Article:||Strong binding and shrinkage of single and double [bar.K] nuclear systems ([K.sup.-]pp, [K.sup.-]ppn, [K.sup.-][K.sup.-]p and [K.sup.-][K.sup.-]pp)...|