Simultaneous Biosensing with Quartz Crystal Microbalance with a

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Simultaneous Biosensing with Quartz Crystal Microbalance with a Dissipation Coupled-Gate Semiconductor Device Toshiya Sakata*,† and Ryushi Fukuda‡ †

Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8656 ‡ Meiwafosis Co., Ltd., 1-14-2 Shinjuku, Shinjuku-ku, Tokyo, Japan 160-0022 ABSTRACT: In this study, we proposed and demonstrated a novel simultaneous analysis system of biosensing by combining a semiconductor-based field effect transistor (FET) with quartz crystal microbalance with a dissipation (QCM-D) monitoring system. Using the combined system, the changes of not only mass and viscoelasticity but also electrical charge for interaction of charged dextran molecules with substrate, recognition of glucose with low molecular weight, and programmed cell death, apoptosis, were simultaneously and quantitatively monitored in a label-free and real-time manner. The combined system will give more detailed information of biomolecule/substrate interface for development of new biomaterial.

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dissipation factor, together with the more commonly measured frequency shift corresponding to mass change based on biomolecular recognition events such as protein adsorption and antibody−antigen reactions.14−17 Combined dissipation and frequency-shift measurements using the QCM provide information about the structure and dynamic viscoelastic properties of surface-adsorbed protein layers. It is very valuable to analyze simultaneously biocompatibility and adsorption between biomolecule and material for development of biomaterial and so on. However, we need to investigate more factors such as electrical properties as well as mechanical ones such as mass and viscoelasticity in order to analyze the adsorption mechanism, which is so closely related to not only specific binding such as DNA hybridization or antigen− antibody reaction but also electrostatic interaction based on molecular charge with material in itself. Therefore, an additional function of electrical measurement for the QCM-D system will be valuable for design and development of biointerface not to mention a simple way to combine their systems. In this paper, we report the combined system for simultaneous measurement of ion charge, mass, and viscoelasticity changes enable to estimate interaction between biomolecule and material. For this purpose, we develop a FETcoupled QCM-D system and investigate adsorption of dextran molecules with positive or negative charges, specific binding of glucose with low molecular weight with phenylboronic acids, and programmed cell death, apoptosis using the combined system.

iochips/biosensors with various principles have been proposed in the fields of clinical diagnosis, pharmaceutical discovery, environment monitoring, and food safety. They are available for rapid and simple screening of components in samples by utilizing miniaturized systems. The principles of detection methods are manifold: for example, based on fluorescence, chemiluminescence, mass and viscoelasticity, refractive index, electrical charge, and so on.1−6 We have been investigating a new approach to analyze simultaneously ion charge, mass, and viscoelasticity changes by combining a semiconductor-based field effect transistor (FET) with quartz crystal microbalance with a dissipation (QCM-D) system. Recently, a FET biosensor is being studied and developed to apply for clinical diagnosis, drug discovery, tissue engineering, and so on.7−13 Since the FET biosensor can detect molecular recognition events accompanied by charge density changes without labeled materials and be easily arrayed by use of the conventional semiconductor processes in order to measure multisamples, the platform based on FET biosensor is suitable for a simple and cost-effective system for chip-based diagnosis. The down-sizing of the system is significant for personalized medicine at home. Moreover, the electrical signals based on FET devices result in direct and quantitative analyses of biosamples. Electrical charges of ions or biomolecules interact electrostatically with electrons at the channel of the semiconductor device. Therefore, ionic behaviors based on biological phenomena can be directly detected using semiconductor devices. Most of the biological phenomena in vivo are closely related to charged mediums, for example, such as DNA molecules with negative charges based on phosphate groups and ions (potassium, sodium, and so on) through ion channel at cell membrane keeping homeostasis. In about a few decades, the QCM has been developed allowing precise, time-resolved measurements of the energy © 2013 American Chemical Society

Received: February 12, 2013 Accepted: May 13, 2013 Published: May 13, 2013 5796

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the difference of VG−ID characteristics at a constant ID of 1 mA. The time course of the surface potential at the gate surface was monitored using a circuit19 with which the potential change at the interface between an aqueous solution and the gate insulator can be read out directly at a constant ID. In the present study, the drain voltage VD and the ID were set to be 2.5 V and 1 mA, respectively. QCM-D Monitoring. The QCM sensors used in this study were 14 mm diameter discs and optically polished AT-cut quartz crystals with Au coating (10 mm diameter) on both sides. Sensors were operated at a fundamental frequency of 4.95 ( f 0) MHz. Frequency at multiple overtones was measured simultaneously. In this study, the results shown were the normalized frequencies calculated from the seventh overtone ( f 7/7). Before each measurement, each measurement solution was passed through the QCM flow module for 30 min to obtain a stable baseline. Adhesion Monitoring of Biomolecules Using the Combined System. Two types of dextran were prepared to investigate the possibility of simultaneous measurement using the combined system. One was diethylaminoethyl-dextran (DEAE-dextran) (Amersham Biosciences) with positive charges, of which a mean molecular weight (MW) was about 500 000. Another was dextran sulfate sodium salt (Amersham Biosciences) with negative charges, of which a mean MW was about 1 000 000 followed by careful purification. Each dextran was in a distilled water solution at a concentration of 1 mg/mL, respectively, and was introduced onto a sensor substrate in turn. The Au sensor surface was treated by use of UV ozone cleaner (MEIWAFOSIS co., ltd) before measurements. Moreover, glucose (10 mM; Wako) was utilized to estimate adsorption characteristics as a detection example of low molecular weight molecule. Then, mercaptophenyl boronic acid (PBA; Sigma Aldrich) was coated on the Au electrode as a self-assembled monolayer (SAM) membrane. The measurements of these molecules were carried out under a constant flow rate of 100 μL/min and at 25 °C. Cell Sensing Using the Combined System. HeLa cells were cultured on the cell culture dish at 37 °C and 5% CO2 in the incubator system. After the preculture, HeLa cells were seeded on the Au electrode of the combined system and cultured on it under the adequate cell culture condition for 1 day in the incubator. After that, the Au electrode with HeLa cells was set up in the fluidic chamber of the QCM-D system at 37 °C. Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) (+1% FBS) was used as culture medium. The number of seeded cells was about 1 × 105 cells/mL for all the experiments. The simultaneous measurement of the combined system was performed at 37 °C and flow rate of 100 μL/min in the fluidic chamber of the QCM-D system. After confirming that signal traces almost become steady, the changes of surface potential, frequency, and dissipation were monitored using the combined system. We set the standard of signal drift less than a few mV per 1 h as baseline of steady potential. The apoptosis was initiated by injecting TNF related apoptosis induction ligand (TRAIL, 500 ng/mL).

EXPERIMENTAL SECTION Structural Concept of a Combined System. In order to combine the FET with the QCM-D system, we have utilized the device structure of extended-gate FET (EG-FET).18 In case of EG-FET, gate electrode is extended from metal electrode of metal oxide semiconductor (MOS) FET, as shown in Figure 1.

Figure 1. Schematic illustration of combined system composed of QCM-D and FET. The structure of extended-gate FET was utilized for this combined system because an Au substrate could be a common electrode.

In this study, p-type Si was used forming an n-channel. Therefore, various kinds of materials and structures can be selected for gate electrode of EG-FET. In this study, an Au electrode prepared for the QCM-D system was used as gate electrode of EG-FET, as shown in Figure 1. The Au electrode was connected with MOS-FET and set up in the fluidic chamber of the QCM-D system, in which the reference electrode was embeded for EG-FET measurement. We have utilized the commercially available electrical module developed by Q-Sense technology (Sweden) in order to have contact with the gate electrode of the FET device. The reference electrode was composed of AgCl-coated Ag in oversaturated KCl solution, which was electrically connected with measurement solution through an agar-based salt bridge or porous glass. The combined system had one common electrode composed of a Au thin film, which was spattered together with Cr on quartz crystal of SiO2. The thickness of Au and Cr film was about 50 and 30 nm, respectively. Electrical Measurement Using FET. The detection principle of FET sensor is based on the potentiometric detection of charge density changes at the gate electrode, on which specific binding between target and probe molecules is made for molecular recognition. Basically, ionic or molecular charges at the gate interact electrostatically with electrons in silicon crystal through the thin gate insulator and induce electrical signals by the field effect, resulting in the source and drain current (ID) change at the channel (Figure 1). The electrical characteristics of FET sensor such as the gate voltage (VG)−drain current (ID) characteristics and the surface potential at the gate surface were measured in a phosphate buffer solution (0.025 M Na2HPO4 and 0.025 M KH2PO4, pH 6.86, Wako) using a semiconductor parameter analyzer (B1500A, Agilent) and a real-time potentiometric analyzer (U2723A, Agilent), respectively. As the basic electrical characteristic, the threshold voltage shift ΔVT was defined as

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RESULTS AND DISCUSSION Figure 2 shows the results of simultaneous adhesion monitoring of different dextran molecules by use of the combined system. First, the real-time monitoring of frequency and dissipation was demonstrated using the principle of QCM-D in the combined system, as shown in Figure 2a. The frequency change shows 5797

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charged dextran sulfate attached and contacted directly with it. Since the FET device was very sensitive to charges of contacted molecules with the electrode, the contacts would be directly reflected as electrical signals in Figure 2b. In this case, the electrical signals should show unstable behaviors, if the attachment between dextran sulfate and Au electrode was not firm. This may also contribute to buffer conditions such as pH, ion strength, and so on. Moreover, the electrical signals became stable after the introduction of DEAE-dextran. This would be because positively charged DEAE-dextran caused stable binding with dextran sulfate resulting in the stable output as shown in Figure 2b. In particular, this result will give the principle which guides one to coat polymer membranes on various substrates for an alternate soaking process based on electrostatic interaction. The data based on QCM-D introduce some analyses such as mass and thickness of attached dextran membranes. From Figure 2a, the changes of mass and thickness of each attached dextran molecule were calculated as shown in Table 1. Table 1 dextran sulfate

Figure 2. Real-time monitoring of different dextran molecules using the combined system composed of QCM-D (A) and FET (B). Frequency, dissipation, and surface potential shifts were continuously monitored at room temperature and at flow rate of 100 μL/min until around 1500 s. Dextran sulfate and DEAE-dextran were introduced onto the common Au electrode in turn through the fluidic chamber. The result of QCM-D was the normalized frequencies calculated from seventh overtone (35 MHz) for fundamental frequency. The QCM-D measurements were evaluated using an adequate overtone in this study, which various targets such as low molecules to polymers including cell attachment or detachment could be detected. On the other hand, the result of FET was measured at a constant drain current of 1 mA, drain voltage of 2.5 V, and gate voltage of 0 V and showed surface potential instead of source voltage as output according to the circuit shown in the previous work.19

mass (ng/cm2) thickness (nm) charge density change based on QCM (C/m2) charge density change based on FET (C/m2)

DEAEdextran

255 2.4 1.6

155 1.5 1

1.3 × 10−4

7.3 × 10−5

Moreover, these calculated data can show the number of charges for negatively charged dextran sulfate and positively charged DEAE-dextran considering molecular weights (1 000 000 for dextran sulfate and 500 000 for DEAE dextran). In this case, the dextran sulfate membrane with two negative charges for monosaccharide included 9.8 × 1018 charges/m2 resulting in about 1.6 × 10 °C/m2, while the DEAE-dexran membrane with three positive charges for tetrasaccharide did 6.3 × 1018 charges/m2 resulting in about 1.0 × 10 °C/m2. The elementary charge has a measured value of approximately 1.6 × 10−19 coulombs (C). On the other hand, the number of ion charges could be calculated from the surface potential changes using the FET device. The interaction between dextran molecules and electrode could be directly transduced into electrical signal using the FET device. The change of surface charge density could be detected as a shift of the surface potential of the FET. The surface potential shift after the attachment of dextran molecules, ΔVFET, can be expressed in eq 1, where ΔQdextran is the charge per unit area of the dextran membranes and Ci is the gate capacitance per unit area.

that each dextran was adsorbed on the Au electrode by the introduction of them. The attachment of dextran sulfate was as much amount of frequency shift as that of DEAE-dextran, while dissipation changes were different for each dextran membrane. The first attachment of dextran sulfate indicates stiff adsorption on the Au electrode but DEAE-dextran with positive charges was electrostatically adsorbed with negatively charged dextran sulfate and seemed to swing on it. Thus, QCM-D shows the structural behavior of adsorbed sample molecules on substrates. However, the electrical information of samples for adsorption cannot be obtained from these detected factors. Figure 2b shows the real-time monitoring of surface charge change due to attachment of different dextran molecules by use of the principle of FET in the combined system. The FET sensing was simultaneously performed with QCM-D monitoring shown in Figure 2a. The surface potential after the introduction of dextran sulfate on the Au electrode decreased by the amount of about 290 mV due to negative charges. Moreover, the introduction of DEAE-dextran induced the positive shift of about 180 mV because of positive charges. Therefore, the sample information on molecular charges as well as mass and viscoelasticity could be obtained by the combined system. In this case, signal noises were found on the curve shown in Figure 2b after the introduction of dextran sulfate. Actually, after dextran sulfate was first put onto the Au electrode, negatively

ΔVFET = ΔQ dextran /C i

(1)

Since ΔVFET = 3.0 × 10 mV for attachment of dextran sulfate and Ci = 4.3 × 10−4 F/m2 for the FET, the amount of charges increasing after attachment of dextran sulfate was calculated to be 1.3 × 10−4 C/m2. Similarly, the amount of charge changes for attachment of DEAE-dextran could be calculated as 7.3 × 10−5 C/m2 considering ΔVFET = 1.7 × 102 mV. The charge densities based on FET measurement were smaller by about four to five digits than those based on QCM measurement. This seems to be because the FET signals indicate the potential changes caused by contact charges which dextran molecules attached directly to the Au gate electrode or degree of 2

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molecular weight molecules can be analyzed clearly and conveniently by use of the combined system. Furthermore, we have performed the real-time monitoring of program cell death, apoptosis as one of the cell functions using the combined system, which has not been previously monitored by QCM-D method. When apoptosis is induced by ligand proteins such as TRAIL, volume reduction of cells is observed accompanied by DNA fragmentation, chromatin concentration, and so on in them resulting in fragmentation of cells. Figure 4

electrolytic dissociation based on equilibrium reaction under the constant pH, although QCM-D responded to the mass change of membrane with thickness. The conventional QCM-D system may not be suited for detecting low molecular weight molecules such as glucose (M.W. 180). On the other hand, the FET system can detect ionic charges for even low molecular weight molecules. Figure 3

Figure 3. Real-time monitoring of glucose binding with phenylboronic acid using the combined system composed of QCM-D (A) and FET (B). Frequency, dissipation, and surface potential shifts were continuously monitored at room temperature and at flow rate of 100 μL/min until around 5 min. Glucose of 10 mM was introduced onto the common Au electrode with phenylboronic acid monolayer at about 1.5 min through the fluidic chamber.

Figure 4. Real-time monitoring of programmed cell death, apoptosis using the combined system composed of QCM-D (A) and FET (B). Frequency, dissipation, and surface potential shifts were continuously monitored at 37 °C and at flow rate of 100 μL/min until around 2 h. TRAIL as apoptosis induction ligand of 500 ng/mL was introduced onto the common Au electrode with HeLa cells through the fluidic chamber.

shows the real-time monitoring of glucose using the combined system. In order to bind glucose molecule specifically, PBAbased SAM membrane was prepared on the common Au surface of the combined system. PBA was expected to react with glucose by diol-binding on the substrate resulting in the induction of negative charge in PBA.20 As shown in Figure 3a, the frequency and dissipation of the QCM-D system changed after the introduction of glucose onto the PBA-coated Au substrate, although the change seemed to be small. Moreover, the surface potential of PBA-coated Au electrode shifted largely to the negative direction after the introduction of glucose (Figure 3b). The electrical signal of FET will show large changes even for smaller size of cation or anion when they have electrical charges. The back of the signal shift after each peak for frequency, dissipation, and surface potential might be based on equilibrium reaction between glucose and phenylboronic acid in flowing buffer solution with glucose at the constant rate of 100 μL/min. In particular, the difference of stabilization for FET and QCM response would depend on the change of charges based on equilibrium reaction at the electrode/solution interface other than that of mere adsorption, because the FET device was very sensitive to charge density changes such as pH variation. Therefore, the adhesion characteristics of low

shows the real-time monitoring of apoptosis using the combined system. When the apoptosis induction ligand of TRAIL was introduced into HeLa cells cultured on the Au surface, the QCM-D system showed the signal changes at a few steps for the frequency and dissipation changes, as shown in Figure 4a. After the addition of TRAIL, apoptosis occurred for almost cultured cells to about 1 h, as shown in Figure 5. Actually, the frequency and dissipation changed drastically at around 1 h. When apoptosis of HeLa cells occurred on the Au substrate, culture medium or protein from cells would flow in the gap between detached cell and substrate. In this case, the inflow of culture medium caused the increase of mass on the Au surface showing the decrease of frequency. At the same time, the attached cells would swing connecting with the Au on the way of apoptosis, resulting in the increase of dissipation, and subsequently be removed from the Au surface after apoptosis completed. When their cells were detaching from the Au substrate, the dissipation decreased while the frequency increased drastically. After almost every cell was detached from the Au electrode, some kinds of proteins in culture medium might have been attached on it or adhesion molecules 5799

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Drs. Michael Rodal, Peter Svensson, Fredrik Pettersson and Jennie Wikström of Biolin Scientific in Sweden for their help and useful discussions. A part of this study was supported by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology (JST).

Figure 5. Microscopic observation of HeLa cells on the common Au electrode of the combined system. HeLa cells were cultured normally on it from the previous day to experiments. After the addition of TRAIL at 0 h, almost all the cells had apoptosis occurring at 1 h resulting in volume reduction and fragmentation.

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REFERENCES

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at the cell membrane would have been remained on it. As a result of that, the frequency signal would not be returned completely to the initial point and the dissipation signal would have fallen to less than that of the beginning of the work out because the attached cells before apoptosis had larger viscoelasticity than the remaining proteins on the Au surface. Simultaneously, Figure 4b shows the real-time monitoring of apoptosis by use of FET in the combined system. The surface potential of FET increased drastically at around 0.7 h after the introduction of TRAIL for apoptosis induction. This positive shift of surface potential may show an increase of positive charges based on K+ at the interface between cells and electrode, as the previous work has shown the possibility of H2O, K+, and Cl− release through the cell membrane before shrinkage of cells in the early stage of apoptosis.21 At the same time, negative charges based on sialic acids at the cell membrane should be detached from substrates after apoptosis, as shown in Figure 5. In short, this positive shift indicates the increase of positive charges based on potassium ion release and the decrease of negative charges based on detachment of sialic acids at the cell membrane due to apoptosis. Thus, the combined system with the different detection principles is available for the simultaneous analysis of complex biofunctions and the development of biomaterials considering the interaction between materials and biological phenomena.

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CONCLUSIONS The above results demonstrate that the combined system composed of QCM-D and FET gives simultaneously some information of biological and chemical events and proceeds further to biofunctional analysis. By combining different sensing technologies, we can use a common electrode, contributing to reduction of cost and trouble, and analyze bio- and chemical functions in a short time measurement. To analyze simultaneously, some factors for interaction between material and biomolecule will help one to design biomaterial with a complex biointerface and understand biological phenomena on substrates. Therefore, the platform based on the QCM-D-FET system is suitable for a label-free, cost-effective, and quantitative detection system for biofunctional analysis in environment, food, and clinical research. 5800

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