Further development of efficient drug-delivery systems for human health care urgently requires new sensitive, selective, and time-domain biosensors for detection of different elements and biological analytes in physiological environments. However, the latter are small and the element or analyte of interest may only be present in relatively small concentrations. In general, nanostructures with large surface-area-to-volume ratios comprise elements with high sensitivity to surface effects and, hence, are suitable for use in such sensors.
Many types of biosensors exist for use in physiological environments. Of these, the potentiometric measurement technique (where no current flow is needed during measurements) using zinc oxide (ZnO) is interesting and applicable, because current flow could potentially damage biological environments and systems. ZnO is biosafe and biocompatible. In addition, it is a semiconductor with photonic properties of potential benefit to biophotonics: it emits UV and visible emission that covers the entire visible spectrum (i.e., it emits white light). In addition, ZnO exhibits excellent electrochemical activity and electron-transfer properties. More importantly, in nanostructure form, it can be grown on any substrate, either crystalline or amorphous, at relatively low temperatures.
We used ZnO nanorods (NRs), grown on the tip of a submicrometer glass pipette at a temperature of <100°C (see Figure 1), as ion- and analyte-sensitive sensors (after proper functionalization) and as a delivery system for efficient photodynamic therapy (PDT) to cancerous cells.1–3 We performed measurements using the potentiometric response of the ZnO NRs-based electrode versus a silver/silver chloride electrode fabricated on the tip of a similar pipette. We first functionalized the working electrode with a proper-selectivity membrane or enzymes. The former allows a specific ion type to pass and accumulate, while the latter enhance a specific electrochemical activity. In both cases, a potentiometric effect is measurable.2 We mechanically manipulated the two electrodes inside single cells. For PDT, we dissolved powdered protoporphryrin in methanol and N, N-dimethylformamide in a 1:1 ratio. We manually dipped the ZnO NR-coated tips three times into this solution. After each dip, the tip was allowed to dry at room temperature. We also used a submicrometer glass pipette covered with bare grown ZnO NRs as a reference PDT device to allow separation of the contribution to the fluorescence effect caused by the NRs and the photosynthesized element. We used these bare and conjugated ZnO NR devices as a local PDT intracellular-photosensitizer delivery system for breast-cancer treatment.
Figure 1. Scanning-electron-microscope images showing (a) a glass pipette at low magnification and (b) zinc oxide (ZnO) nanorods (NRs) on the pipette's tip used as a working electrode, at high resolution. (c) High-resolution transmission-electron-microscope ZnO NR image, revealing the NRs' crystalline quality. (inset) Selected-area diffraction pattern.
We measured concentrations of the most important metallic ions—including calcium, magnesium, sodium, and potassium ions—in different types of cells. The results were consistent with measurements using other, indirect techniques. In addition, using the same electrode but replacing the membrane functionalization by enzymes, we measured concentrations of other biological analytes. While we measured glucose, cholesterol, and uric acid traces very accurately in extracellular environments, glucose was also detected inside single cells.2 Figure 2(a) and (b) shows a schematic diagram of the measurement principle. Figure 2(c) shows adipocytes cells under potentiometric measurement, while Figure 2(d) displays the calcium concentration versus that in the buffer solution for the configuration shown in Figure 2(a). Moreover, triggering of cell necrosis/apoptosis was possible for different types of cancerous cells.
Figure 2. Principle of intracellular measurements. (a) Partial insertion of the functionalized ZnO NRs. (b) Insertion of all functionalized ZnO NRs. Ag/AgCl: Silver/silver chloride. (c) Micrograph of adipocyte cell during potentiometric measurement. (d) Measured calcium-ion concentration for the setup in panel (a) versus calcium-ion concentration in the buffer solution. EMF: Electromotive force. No change was seen when the same experiment was repeated for the configuration in (b).
Our main effort has focused on making the tip geometry small enough. Extremely sharp (submicrometer dimensions) and long (>10μm) tips are the basic requirement for our intracellular PDT device. Such devices should have the ability to bend and gently penetrate the flexible cell membrane. These properties are offered by ZnO NRs. Although the technique is invasive, the penetrated cells did not appear to be affected by the invasion. We studied the cancer cells using an inverted fluorescence microscope (Zeiss). We noticed that localized cell necrosis/apoptosis is achieved when ZnO NRs functionalized with a photosensitizer are excited with light,3 i.e., singlet oxygen is created in the cell. Figure 3 shows micrographs taken at different stages of PDT treatment. Cell necrosis/apoptosis was seen for exposures of up to approximately 10 minutes.3
Figure 3. Digital images taken during intracellular photodynamic-therapy measurements. (top left) Fluorescence images of protoporphyrin IX dimethyl ester (PPDME)-conjugated ZnO NRs. (top right) PPDME-conjugated ZnO NR tip inside a cancer cell. (bottom left) Cancer cell under necrosis/apoptosis with intracellular penetration of the conjugated tip. (bottom right) Tip after cancer-cell necrosis/apoptosis.
In summary, with the aim to develop possible future physiological-environment biosensors suitable for human health care and efficient drug delivery, we developed and tested ZnO NR-based sensors. The results showed that our newly developed technique offers promise for selective, sensitive, and time-domain sensors suitable for intracellular-ion and biological-analyte analysis. It can be further extended to use the excellent optical properties of ZnO for development of an efficient PDT system for localized cancerous-cell treatment.
Magnus Willander, Omer Nur
Department of Science and Technology (ITN), Linkoping University
Magnus Willander is a full professor. His research focuses on experimental and theoretical nanoscience using solid and soft materials. He has published 900 scientific articles and eight books.
Omer Nur is an associate professor. His research interests focus on device physics and technology. He has published approximately 170 articles.
1. M. H. Asif, A. Fulati, O. Nur, M. Willander, C. Brännmark, P. Strålfors, S. I. Börjesson, F. Elinder, Functionalized zinc oxide nanorod with ionophore-membrane coating as an intracellular Ca2+ selective sensor, Appl. Phys. Lett
. 95, no. 2, pp. 023703, 2009. doi:10.1063/1.3176441
2. M. H. Asif, S. M. U. Ali, O. Nur, M. Willander, C. Brännmark, P. Strålfors, U. H. Englund, F. Elinder, B. Danielsson, Functionalized ZnO-nanorod-based selective electrochemical sensor for intracellular glucose, Biosens. Bioelectron
. 25, no. 10, pp. 2205-2211, 2010. doi:10.1016/j.bios.2010.02.025
3. S. Kishwar, M. H. Asif, O. Nur, M. Willander, P.-O. Larsson, Intracellular ZnO nanorods conjugated with protoporphyrin for local mediated photochemistry and efficient treatment of single cancer cell, Nanoscale Res. Lett
. 5, no. 10, pp. 1669-1674, 2010. doi:10.1007/s11671-010-9693-z