Quartz Crystal Microbalance as a Sensor for Biochemical and Medical Research

F. N. DULTSEVInstitute of Semiconductor Physics

Siberian Branch of the Russian Academy of SciencesLavrentiev ave., 13, Novosibirsk

RUSSIAfdultsev@thermo.isp.nsc.ru

Abstract: - A method based on the use of quartz resonator as an active element is proposed. It is demonstrated experimentally, with the ensemble of test objects having the characteristic size of 20-30 nm, that this method allows one to determine nanoparticle size distribution (in particular, for viruses). Application of threshold mode enabled us to enhance the sensitivity of the method. The proposed procedure may become a good alternative for existing methods, such as AFM or optical detection of biological objects, and win broad application both in laboratory research and in biosensor applications, including tests for the purposes of diagnostics, drug discovery, and therapy.

Key-Words: - Quartz Crystal Microbalance, Biosensor, Rupture Event, Virus, Recognition

IntroductionInteraction between molecules is a key item for the dynamics of interactions in the system receptor – agent (adsorbent – adsorbate), including biological systems. Molecular interactions are usually considered using thermodynamic approach. However, rupture of bonds formed in these interactions is a dynamic process and thus depends not only chemical affinity but also on the rate of changes of the force applied to the interacting molecules [1]. Investigating biological systems, it is very important to decrease perturbations of the system under investigation but conserve the information content of the system's response to the action. Perturbation may be decreased by excluding the electromagnetic action, for example by using mechanical action to break a bond. This possibility was implemented when Atomic Force Microscopy (AFM) was developed. This method allows one to measure rupture forces for separate particles. One of the disadvantages of AFM is the necessity to localize a particle on the surface before measuring. We propose a new method that does not require such localization; in addition, it is much simpler and cheaper. Our method is based on Quartz Crystal Microbalance (QCM). Quartz generators had been used as microbalance for a long time. The first step was made in 1959; it was demonstrated in [2] that the resonance frequency (fres) of a crystal

depends on the total oscillating mass. The principle of microbalance is frequency shift Δf resulting from the addition of a definite mass (m) on the electrodes or its removal. Sensitivity may reach 1 ng/cm2, which corresponds to a monolayer of adsorbed substance. The second step was made in 1982 by Nomura and Okuhara [3] who demonstrated that stable oscillations may be obtained in a crystal submerged in a liquid. Since then, QCM has become a tool in electrochemistry.The AT-cut quartz resonator is usually used in these measurements. Due to its small temperature coefficient and high Q-factor, it is widely used as a sensitive element in gas sensors, chemical and biological sensors. To enhance sensitivity, resonators made of very thin quartz plates (less than 20 µm) are manufactured. However, there is a limitation connected with temperature drift: the ratio of total oscillating mass to the mass to be measured cannot be better than 109. In order to remove this limitation and increase the sensitivity, it is necessary to perform measurements in a different manner. The third step was made in 2000 when it was shown in [4] how QCM may be used in the active mode.

2 Problem FormulationIt is necessary now to explain the physical

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sense of the phenomenon involving the generation of rupture signal.

All the methods describing oscillations in a quartz crystal may be divided into three groups. The first one involves equivalent circuit model representing the resonator as a branched oscillatory circuit in which the effect of the distributed mass load on impedance changes is taken into account using radiotechnical means [5-7]. The second group uses various 2D and 3D models a priori assigning the distribution of displacements [8, 9]. In the methods of the third group, direct equations of motion are solved to calculate the oscillations in the quartz crystal [10-13]. Finally, the fourth group uses direct 3D modelling by means of finite element method [14-15] allowing one to take into account the effect of crystal anisotropy, piezoeffect and metallization of resonator electrodes.

In our work, we used our own semi-empirical model that provides an analytical description of the interaction of a small particle with the QCM surface. The model does not involve direct calculation of the effects of metallization, anisotropy, piezo-effect etc. on the character of oscillations relying on the basic principles; instead, the values that are difficult to calculate are introduced from the experiment; these values take into account all the effects de facto. To make this method a useful tool for medicine or biology, it is important not to calculate oscillation frequency but to leave it in the free form until an exact measurement is made; thus, more attention may be paid to the parameters of the particles under investigation.

3 Problem SolutionThe above-described limitation may be

removed is QCM is used not only as a sensor but also as an active element. In these measurements, not frequency shift is measured but a sharp acoustic signal generated during particle rupture from the surface under oscillations with increasing amplitude is detected. We succeeded in increasing the sensitivity by 3 orders of magnitude. Measurement diagram is shown in Fig. 1.This arrangement allows us to detect the rupture of a single particle. We demonstrated experimentally that the rupture of a single virus

is reliably detectable [16].

Fig. 1. Set-up for measurements at a fixed frequency (3F+df). G1,G2 are generators, A - lock-in amplifier, PC – computer [4].

However, a virus is rather large particle with the diameter about 100 nm. This arrangement has a shortcoming: the measurements are carried out at the frequency 3F+df, here df depends on many factors, and it is difficult to determine this value beforehand. So, we modified the arrangement as shown in Fig. 2.

Fig. 2. Scheme of the set-up involving analyser.

We propose to work not at a fixed frequency of the chosen harmonics but within a definite band scanning near the resonance value, so that df surely gets into the scanning range. We applied analyzer for this purpose. Using the new arrangement, we obtain a more complete picture of the interaction of a particle with the quartz surface. The results of these measurements are shown in Fig. 3. The measurements were made with a resonator cut as a strip from a round quartz plate along the shear direction; the resonator size was 8 mm x 1.5 mm. One can see in Fig. 3 that mathematical processing is important in this case. B-spline distinguishes the peaks in the

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best manner.

Fig. 3. 3D representation of the experimental acoustic signal recorded using the analyzer. The data relate to Nano Glass Beads (Microspheres-Nanospheres a Corpuscular company, catalogue No. 149015-05).

As our method is based on compensation of binding forces, this procedure allows us to use the same surface to investigate adsorption of molecules bound with different forces. As an example, Fig. 4 shows rupture signals for model nanometer-sized spheres. Glass beads with a size 20-30 nm (size scattered within 25 %) were deposited on the QCM surface.

Fig. 4. The acoustic signal derived from measurements depicted in Fig. 3. Test measurements were carried out using standard nano-sized spheres Nano Glass Beads with declared diameter 20-30 nm (the same as in Fig. 3). Nanoparticle size distribution is shown in the insert.

Thus obtained particle size distribution (shown in the insert) is in good agreement with the declared one. In this form, the procedure may be used to examine nanoparticles of different kinds (e.g. viruses, phages) and to distinguish between different types of particles.

Rupture signals for the vapour of different compounds are shown in Fig. 5. These measurements were made with the quartz crystal coated with porous silicon dioxide. Scanning within a frequency band provides the possibility to avoid the loss of information concerning rupture forces. One can see that different molecules get detached at different amplitudes of quartz surface oscillations.

It is interesting that the shape of peaks for water vapour depends on the concentration, while for nonpolar species (heptane in our case) it does not.

With high water vapour concentrations, two major peaks are observed, while with small concentrations the first peak (at the voltage about 1 V) is almost unseen. This behaviour is likely to be connected with the existence of different forms of physically adsorbed water on the surface; we detect even weakly bound water. On the basis of the positions of these peaks, we estimated the size of the formed clusters. Our measurements suggest that the clusters are composed of 3-4 water molecules The formation of the clusters of this size is quire possible, provided that the surface not tightly coated with OH groups. The authors of [17] made estimations using semi-empirical methods (MNDO and AM1) and concluded that the surface cluster is composed of 2-4 water molecules; this is in good agreement with our results. The energy of water bonding to the surface OH group was calculated to be about 4.3 kcal/mol, which means just physical adsorption.

Changes of the acoustic signal with time at a constant amplitude of quartz surface oscillations (voltage: about 1 V) are shown in Fig. 6. This is a threshold measurement mode [18]. The arrow in the plot indicates the moment when water vapour is admitted. Water vapour was admitted in the pulse regime; the pulse duration was 0.5 s. Dry nitrogen was admitted through channel 1, while a mixture of water vapour (70 %) with nitrogen was admitted through channel 2; the ratio between channels was 100:1. Measurements were performed at Т=20 oC. Inserts in the upper part of Fig. 6 show the data on the intensity of the acoustic signal after combined smoothing using B-spline.

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Fig. 5. Experimental 3D intensity of the acoustic signal for different gases on the same surface: (a) ethanol, 30%; (b) high water vapour concentration (humidity 70%); and (c) heptane, 1%. The temperature is 20 C.

Fig. 6. Response of the system versus time at the constant amplitude (1 V) of the threshold voltage, with the pulsed admission of water vapor into the chamber. The ratio of flows through two channels is 100:1. The temperature is 20 C.

The lower plot shows the signal averaged over frequency span. One can see that the useful signal is well detectable. The signal has very steep edges, so the sensor is fast-operating (it has a high response rate). Operation in the threshold mode allows increasing the number of molecules interacting with the surface within unit time (stimulated adsorption-desorption). Comparing the dependencies recorded using different measurement modes we see that the sensitivity of measurements in the threshold mode is two orders of magnitude higher.

4 Conclusion

Thus, the active mode of quartz resonator operation allowed us to enhance the sensitivity of the method and to determine nanoparticle size distribution, provided that the particles cover not more than 1% of the quartz surface. This allows one to use this method for

detection and separation of biological bodies such as viruses or phages. The procedure proposed for processing experimental data to obtain particle size distribution is demonstrated with test nanoparticles as an example; a good agreement with the declared particle size was achieved. Application of scanning within a frequency band simplified the adjustment procedure and allowed us to obtain a more complete picture of nanoparticle rupture from the surface. Operation in the threshold mode resulted in the additional increase in the sensitivity of the proposed method.

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