Report
ULTRASMALL for Cellular With dimensions in the micrometer-tonanometer range, the nanoscopic optical biochemical sensor offers fast response time and excellent biochemical sensitivity. In the biomedical sciences, the greatest advances in the last two decades have been inspired by the genome project. What comes after deciphering the genetic code? Certainly, one next step is understanding the biochemistry driven by the gene, from the cellular nucleus to its organelles, cytoplasm, and beyond. One important goal is to follow in real time the chemical kinetics and dynamics of the living cell, much of which involves small ions and molecules.
Weihong T a n University of Florida Raoul K o p e l m a n Susan L. R. Barker M i c h a e l T. Miller University of Michigan 606 A
So far, there have only been a couple of approaches to real-time chemical analysis in living mammalian cells—electrochemical microsensors and nanosensors, and fluorescent molecular probes. The latter are based on free molecules, which do not constitute a sensor. The aim has long been to replace such fluorescent molecular probes with fluorescent sensors that are still small enough for noninvasive intracellular chemical analysis and, at the same time, immune to interference from cell ingredients. The miniaturization of biochemical sensors small enough for cellular and subcellular applications has been difficult. Recent advances in microfabrication techniques based on scanning probe microscopy, lithography, and screen printing have enabled the development of many miniaturized analytical devices. In this Report, we discuss a new device called a nanoscopic optical biochemical sensor (NOBS), which has dimensions in the micrometer to nanometer range, fast responsetime,and excellent biochemical sensitivity. A NOBS uses the high biochemical selectivity of optically sensitive dye molecules or biomolecules, such as enzymes, antibodies, DNA molecules, or living cells, to recognize substances of interest in complex media. The sensing component is directly connected with a signal transducer. Biochemical sensors with dimensions in the micrometer to nanometer range have been developed and applied in a wide variety of fields,frombiomedical diagnosis to envi-
Analytical Chemistry News & Features, September 1, 1999
ronmental detection (1-17). Because increasing use of these ultrasmall sensors is expected in the near future, especially for biomedical applications, we will emphasize advances made in intracellular and extracellular biochemical sensors. Optical sensor preparation
The fabrication of structures with dimensions down to the submicrometer range is already well known in standard microelectronics. However, reproducing biochemical sensors with acceptable performance is a highly technical challenge that needs further development. In preparing a NOBS, two basic elements are needed—a miniaturized support and an ultrasmall sensing component. Using near-field optics (NFO) (18-20), a novel nanofabrication technology has been developed for preparing structures. Photonanofabrication, based on NFO, controls the size of the luminescent material grown at the end of a light transmitter, such as an NFO tip (Figure la), by photochemical reactions. These reactions are initiated and driven by an appropriate wavelength of light. The luminescent material is synthesized only in the of light and is "bonded" only to the where light is emitted. The key to photonanofabrication is a near-field photochemical tion in which the electromagnetic of the light source are maDDed by the photochemical process Thus the size of the luminescent is defined
OPTICAL SENSORS
Measurements emitting aperture and is independent of the wavelength of the light used to promote the chemical reaction. The photochemical reaction only occurs in the near-field region, where the photon flux and the absorption cross-section are the highest (1, 16). The NFO-based nanofabrication process is shown in Figure lc. As an example, the near-field photopolymerization process for preparing a submicrometer optical-fiber pH sensor is described (16). The NFO fiber tip is first pretreated by silanizing with a 3-(trimethoxysilyl)propyl methacrylate solution and then placed under nitrogen for 1 h. The silanized tip end is then sensitized with a solution of benzophenone in cyclohexane. After pretreatment, the photopolymerization is controlled by the light emanating from a near-field light source. The size of the light source and the nearfield photon profile control the dimension and shape of the immobilized photoactive polymer The pH sensors are prepared by incorporating a fluoresceinamine derivative, acryloylfluorescein, into an acrylamidemethylene bis(acrylamide) copolymer, which is attached covalently to a silanized NFO tip surface by photopolymerization. The sensitized end of the optical fiber is placed in contact with the monomer solution, which ensures size control of the formed polymer. The optical beam from the NFO tip initiates the polymerization process. The rate and size of polymer formation are controlled by visually monitoring the distal
tip through a microscope. This process enables the incorporation of pH-sensitive dye molecules covalently bonded to the NFO tip surface, yielding the nanometer-sized biochemical sensors (1,16). Submicrometer structures at the end of an NFO probe can thus be produced by using photonanofabrication (Figure 2a). The scanning electron image shows that the polymer grown on the NFO probe has a different shape from the probe itself. The photonanofabrication technique has been generalized by using fluorescent dye or fluorescent dextran-doped polymers through photopolymerization (21). This makes it possible to further miniaturize a NOBS with multiple biochemical sensitivities by multiple-step nanofabrication T) r o p f* s s f* ^
Other methods that have been used to prepare micrometer-sized sensors are less controllable and do not produce well-defined sensing structures. For example, many sensors have been prepared using a simple dipping method (5,10,22-30.. Optical-fiber probes can be made in the submicrometer to the tens of micrometers range by etching with hydrofluoric acid or by using a laserbased puller. Submicrometer fiber tips also have been prepared using conventional fusion splicer technology (11)
Figure 1 . (a) Unmodified optical-fiber tip and (b) the new "supertip". (c) Photonanofabrication process by near-field optics.
Once the fiber probes are ready, a variety of simple methods are used to prepare a NOBS (2, 5, 21-30). Two parameters exist for controlling the sensor size—the size of tiie optical-fiber probe and the posi-
Analytical Chemistry News & Features, September 1, 1999 6 0 7 A
Report
Figure 2. (a) Scanning electron micrograph of a pH nanosensor probe. (b) Microphotograph of an NFO biochemical sensor prepared by photonanofabrication. The bright tip area shows that sensing molecules are attached to the tip surface.
tioning of the probe during preparation. Usually, a microscopic positioning device is used to physically control the preparation of the sensing element. However, a micrometer-to-submicrometer NOBS has been created using sol-gel matrixes and polyvinyl chloride by simple dipping (10,11). Intracellular ion sensors
Nanometer pH sensors with subwavelength dimensions have been prepared with various dye molecules (1,10,11,16, 21,26). The bright point of light at the end of the fiber probe in Figure 2b is the fluorescence from a dye embedded in the copolymer covalently bonded to the activated fiber surface. To test the applicability of the pH sensor for measurements in small volumes, the sensors were positioned in porous polycarbonate membrane holes 608 A
(0.02-20 um)filledwith buffer. Eight different buffers were used to progressively change the pH in each hole from 4 to 9 and then back to 4. The covalent bonding of the sensing elements means that the analytes have immediate access to the dye on the sensor tip, which gives the NFO sensors the fastest response times among reported opticalfiber sensors, for example
