Optical Nanosensors for Detecting Proteins and Biomarkers in Individual Living Cells

Tuan Vo-Dinh

Summary

Recently, nanotechnology has been revolutionizing important areas in molecular biology and medicine, especially diagnostics and therapy at the molecular and cellular levels. The combination of nanotechnology, biology, advanced materials, and photonics opens up the possibility of detecting and manipulating atoms and molecules using nanodevices. This capability has the potential for a wide variety of medical uses at the cellular level. One of the most recent technological advances has been in the area of nanosensors. This chapter describes the principle of optical nanosensors, their development, and their applications for in vivo analysis of proteins and biomarkers in individual living cells. Nanosensors were fabricated with optical fibers pulled down to tips with distal ends in nanoscale dimensions. Nanosensors with immobilized bioreceptor probes (e.g., antibodies, enzyme substrate) that are selective to target analyte molecules are also referred to as nanobiosensors. Laser light is launched into the fiber, and the resulting evanescent field at the tip of the fiber is used to excite target molecules bound to the antibody molecules. A photometric detection system is used to detect the optical signal (e.g., fluorescence) originating from the analyte molecules or from the analyte-bioreceptor reaction.

Key Words: Nanosensor; nanoprobe; nanotechnology; biosensor; antibody; single cell; benzopyrene tetrol; cancer.

1. Introduction

Minimally invasive analysis of proteins and related cellular signaling pathways inside single intact cells is becoming increasingly important because cells in a population respond asynchronously to external stimuli. There is a need to further our understanding of basic cellular signaling processes in order to obtain new information that is not available from population-averaged cellular measurements. A further advantage of living-cell analysis is that it allows us to

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press Inc., Totowa, NJ

understand the exact pathways by which signaling pathways move through the architecture of the cell. Not only is there a need to resolve such measurements temporally; there is also a need to resolve them spatially. For these reasons, continued progress in cellular physiology requires new protein measurement strategies that can be applied to individual cells with great temporal and spatial resolution.

Advances in nanotechnology and photonics have recently led to a new generation of devices for probing the cellular machinery, elucidating intimate life processes occurring at the molecular level that were heretofore invisible to human inquiry (1). Recent advances in nanotechnology have led to the development of biosensor devices having nanoscale dimensions; such sizes make them suitable for probing biomolecules such as proteins inside individual living single cells. These nanotools could provide new information that could greatly improve our understanding of cellular function, thereby revolutionizing cell biology.

Fiberoptic sensors provide useful tools for remote in situ monitoring. Nanoscale fiberoptic sensors would be suitable for sensing intracellular/inter-cellular physiological and biological parameters in microenvironments. A wide variety of fiberoptic chemical sensors and biosensors have been developed in our laboratory for environmental and biochemical monitoring (2-9). Submicron fibers have been developed for use in near-field optics (10,11). Tapered fibers with submicron tip diameters between 20 and 500 nm have also been developed for near-field scanning optical microscopy (NSOM). NSOM was used to achieve subwavelength 100-nm spatial resolution in Raman detection (12,13). Tan et al. (14,15) have developed and used chemical nanosensors to perform measurements of calcium and nitric oxide, among other physico-chemicals in single cells. Vo-Dinh et al. have developed nanosensors with antibody probes (16-23) and enzyme substrate-based probes (24) to detect biochemical targets and proteins inside living single cells. This chapter describes the operating principle, instrumentation, protocols, and applications of optical nanosensors.

2. Principle of Biosensors and Nanosensors 2.1. Near-Field Optics and Nanosensors

Nanofibers were originally developed for use in NSOM, which is a technique involving light sources or detectors that are smaller than the wavelength of light (10). The first method developed for performing these experiments was to place a pinhole in front of the detector, thus effectively reducing the detector's size. In a later variation of these pinholes, an excitation probe with dimensions smaller than the wavelength of the light was used for sample inter rogation. Betzig and Chichester (11) developed one such probe capable of obtaining measurements with a spatial resolution of approx 12 nm. The probe was constructed by using a micropipet puller to pull a single-mode optical fiber to a tip diameter of 20 nm and then coating the walls of the fiber with 100 nm of aluminum to confine the excitation radiation to the tip. With this nanoprobe, images of a pattern were reconstructed from a raster scan performed in the illumination mode, with the probe acting as a localized light source.

Near-field microscopy has received great interest resulting from its extremely high spatial resolution (subwavelength) (10). For example, a relatively new method known as near-field surface-enhanced Raman spectroscopy (NF-SERS) has been used for the measurement of single-dye and dye-labeled DNA molecules with a resolution of 100 nm (12,13). In this work, DNA strands labeled with the dye brilliant cresyl blue were spotted onto a SERS-active substrate that was prepared by evaporation of silver on a nanoparticle-coated substrate. The silver-coated nanostructured substrates are capable of inducing the SERS effect, which can enhance the Raman signal of the adsorbate molecules up to 108 times (25). NF-SERS spectra were collected by illuminating the sample using the nanoprobe and detecting the SERS signals using a spectrometer equipped with a charge-coupled device (CCD). Raster scanning the fiber probe over the sample and normalizing for surface topography using Rayleigh scattered light produced a two-dimensional (2D) SERS image of the DNA on the surface of the substrate with subwavelength spatial resolution. Near-field optical microscopy promises to be an area of growing research that could provide an imaging tool for monitoring individual cells and even biological molecules. Single-molecule detection and imaging schemes using nanofibers could open new possibilities in the investigation of the complex biochemical reactions and pathways in biological and cellular systems.

2.2. Biosensor Components

Over the years, new techniques in biosensing have set the stage for great advances in the field of biological research. The two fundamental operating principles of a biosensor are biological recognition and sensing. Therefore, a biosensor can be generally defined as a device that consists of two basic components connected in series: (1) a biological recognition element, often called a bioreceptor; and (2) a transducer (4,18). The biosensor detects molecules and transforms this recognition into another type of signal using a transducer. The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into a measurable effect, such as an electrical signal. A bioreceptor is a biological molecular species (e.g., an antibody, an enzyme, a protein, or a nucleic acid) or a living biological system (e.g., cells, tissue, or whole organisms) that uses

Bioreceptor Probe

Was this article helpful?

0 0

Post a comment