Fundamentals of Molecular Interaction Analyzer QCM

Oscillation method (QCM)

Principle.

A quartz crystal resonator (Also called a crystal oscillator) is that a quartz crystal is cut into very thin plates and metal thin films are attached to both sides. When an AC electric field is applied to each metal thin film, the quartz crystal resonator vibrates at a certain frequency (resonant frequency). When a nanogram of a substance is adsorbed on a metal thin film, the resonance frequency decreases in proportion to the mass of the substance so that it can be used as a microbalance. This method is called the QCM (Quartz Crystal Microbalance) method.The principle of the QCM method is briefly introduced below.

Piezoelectric effect of a quartz crystal.

In some crystals, mechanical strain changes the positional relationship of the atoms in the crystal. As a result, polarization occurs in proportion to the magnitude of the strain. This phenomenon is called the Piezoelectric effect and was discovered by the French physicist Curie brothers in 1880.

On the contrary, applying an electric field to a crystal causes mechanical strain (inverse piezoelectric effect). These phenomena are found in crystals such as quartz crystal, Rochelle salt, and tourmaline. Among them, quartz crystal(quartz; SiO2) is used the most because it is known to have excellent piezoelectric properties, chemical properties, and thermal stability.

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Quartz Crystal Microbalance method.

When metal electrodes are attached to both sides of an extremely thin plate which is cut out of a quartz crystal in a certain crystal direction and an AC electric field is applied to each electrode using an oscillation circuit, it becomes a quartz crystal resonator that vibrates at a certain frequency (resonant frequency). This quartz crystal resonator is used as a reference frequency for communication equipment such as quartz watches and mobile phones because it is easy to use and provides high-precision and stable frequencies.

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There are various types of quartz crystal resonators. A quartz crystal resonator whose electrodes are formed on both sides of a quartz crystal plate thin film that is cut in an angle called AT-cut is called Thickness-shear-mode resonator and shakes in a horizontal direction to the quartz crystal surface.

The frequency of the quartz crystal resonator is determined by the thickness of the quartz crystal. By using a thin quartz crystal plate, a quartz crystal resonator that vibrates at a higher frequency can be obtained. It was reported in the 1950s that the frequency of the quartz crystal plate changed according to the mass of the substances on the electrode.

The relationship between variation of frequency and the mass of adhering substance is expressed by the Sauerbrey equation. The frequency decreases as the mass of adhering substance increases, and the frequency increases as the mass of adhering substance decreases. The quartz crystal microbalance method (QCM method) uses this phenomenon to measure the mass change of a substance on the electrode by detecting the frequency change of the quartz crystal resonator.

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Quartz crystal resonators used to be used for primarily gas-phase measurements.

The reasons are (1) the quartz crystal plate cannot oscillate because its vibration is strongly damped in a viscous environment such as the liquid phase, and (2) the structure of a quartz crystal resonator is a kind of condenser whose metal thin films sandwich a quartz crystal which is an insulator, and both electrodes are energized in water. However, in 1981, it was shown that oscillation could be achieved even in the liquid phase. Improvements in the oscillation circuit and in the coating of one side of the quartz crystal resonator to prevent contact with the liquid phase made it possible to use the quartz crystal resonator in the liquid phase.

These developments have led to a significant increase in the use of QCM in water. Over the past 10 years, they have been used in a variety of fields, including chemistry, biochemistry, and microbiology.

QCM as a Molecular Interaction Measuring device.

The Sauerbrey equation contains the fundamental frequency F0 of the quartz crystal resonator in the right-hand side.

This indicates that the fundamental frequency of the quartz crystal resonator becomes higher, the value of the detected frequency change becomes larger in proportion to the square of the frequency, in other words, mass detection sensitivity becomes higher.

We succeeded in commercializing the world's first QCM device using a quartz crystal resonator with 27 MHz high-frequency fundamental frequency, which had been difficult to oscillate in an aqueous solution.

In the case of a quartz crystal resonator with a fundamental frequency of 27 MHz used in the sensors of AFFINIX series, it has been confirmed both theoretically and experimentally that a frequency decrease of 1 Hz indicates 0.62 ng/cm2 of mass increase on the sensor surface.

This means that the detection signal is 29 times and 9 times higher than the 5 MHz and 9 MHz quartz crystal resonators respectively found in the market.

The use of high-frequency QCM improves the detection sensitivity, which enables to detect changes in frequency over time in the interaction of various molecules (Nucleic acids, peptides, proteins, sugar chains, lipid monolayers) in living organisms which had been difficult to observe because the amount of change is small.

QCM products normally collect frequency data every second to monitor changes in mass in real time.It is very important to be able to monitor the adsorption of substances on the surface in real time, and information such as the adsorption rate of substances can also be obtained.

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The features of QCM devices AFFINIX series are as follows.

The amount of adsorption of the nanogram level on the sensor surface can be quantified.
Interaction of biomolecules such as proteins can be detected in near-native states without being labeled.
The adsorption reaction can be measured in real time.

For the AFFINIX series, the number of frequency decreases because the mass increases in the binding reaction, and the number of frequency increases in the decomposition reaction because the mass decreases.This indicates that AFFINIX series can be used can be used to adsorb molecules such as proteins on solid surfaces such as gold and SiO2, to quantify the adsorption amount of high polymer thin films, and to monitor their dissociation and decomposition.


When quantifying the interaction between two molecules, the adsorption amount can be monitored and measured by immobilizing one of the molecules on the surface of the sensor and by adding the other molecule to the solution.

Molecules that are immobilized on the surface of the sensor are called host molecules. Molecules that are added to the solution and bind to host molecules are called guest molecules.is added to a host molecule immobilized substrate, it initiates the binding reaction to the host molecule.

The guest molecules adsorption amount is concentration-dependent. The dissociation constant (Kd Value), which indicates the binding strength between the host molecule and guest molecule, can be determined by analyzing the equilibrium value of each concentration.Also, since the binding reaction can be monitored in real time, the parameter binding rate(kon) and dissociation rate (koff) of reaction rate can be determined by analyzing the adsorption curve at the time of binding. The dissociation constant is the inverse number of the binding constant (Ka). When the dissociation constant is smaller, the affinity between the host and guest molecules is greater.

However, even if it is an interaction with the same dissociation constant value, there is a reaction with fast binding rate and slow dissociation rate, and a reaction with a fast binding rate and a fast dissociation rate. When analyzing the details of intermolecular interactions, it is important to include the binding and dissociation rates.


Experiments to calculate the dissociation constant (binding constant) can be performed by calculating the saturated adsorption amount ⊿ F for each concentration and plotting it as the horizontal axis guest molecule concentration and the vertical axis saturated adsorption amount. If the experiment is conducted correctly, the plot will be a Langmuir-type saturated plot which approaches a certain value as the concentration increases. The optimal combination of Kd and ⊿ Fmax can be derived by curve-fitting the obtained plot using nonlinear regression calculations.

There are two methods for calculating the saturated adsorption amount for each guest molecule concentration. One is a method to determine the equilibrium adsorption amount of each guest molecules concentration by adding a guest sample and repeating the process of adding another guest sample when the adsorption reaches equilibrium. Another is a method of repeating independent measurements adding guest samples with different guest molecule concentrations. Both methods can obtain the equilibrium adsorption amount of each guest molecule concentration. The same value can be obtained in principle.


In a method of repeating independent measurements by adding guest samples with the different guest molecules concentrations, parameter binding rate (kon) and dissociation rate (koff) of the reaction rate can be calculated by fitting the adsorption curve. We offer AQUA software, which allows easy fitting of sensorgrams and calculation of equilibrium constants for analysis of the interaction measurement data of AFFINIX series.

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Fundamentals of Molecular Interaction Analyzer QCM

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