Optochemical nanosensor PEBBLEs: photonic explorers for bioanalysis with biologically localized embedding Sarah M Buck 1 , Yong-Eun Lee Koo 1 , Ed Park 1 , Hao Xu 2 , Martin A Philbert 2 , Murphy A Brasuel 3 and Raoul Kopelman 1 Nanosized photonic explorers for bioanalysis with biologically localized embedding (PEBBLEs) have been created for the intracellular monitoring of small analytes (e.g. H + , Ca 2+ , Mg 2+ , Zn 2+ ,O 2 ,K + , Na + , Cl  , OH and glucose). The probes are based on the inclusion of fluorescent analyte-sensitive indicator dyes and analyte-insensitive reference dyes in a polymer (polyacrylamide, polydecylmethacrylate) or sol-gel (silica, ormosil) nanoparticle. The probes are ratiometric, reversible and protected from interaction with the cellular environment, a quality which is of benefit to the integrity of both the cell and the sensor functionalities. Herein we describe two types of PEBBLE sensors, direct measurement sensors and ion correlation sensors, as well as the use of these PEBBLEs in intracellular sensing. Addresses 1 Department of Chemistry, University of Michigan, Ann Arbor 48109 Michigan, USA 2 Department of Environmental Sciences, University of Michigan, Ann Arbor 48109 Michigan, USA 3 Department of Chemistry, Colorado College, Colorado Springs 80903, Colorado, USA  e-mail: kopelman@umich.edu Current Opinion in Chemical Biology 2004, 8:540–546 This review comes from a themed section on Analytical techniques Edited by Renato Zenobi and Fred Regnier Available online 25th August 2004 1367-5931/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.08.011 Abbreviations PDMA polydecylmethacrylate PEBBLEs photonic explorers for bioanalysis with biologically localized embedding PEG polyethylene glycol Introduction In cellular research, one strives to become a ‘silent observer’, accurately measuring and observing processes without interfering with normal cell behavior. Conven- tional methods for cellular chemical imaging include free (‘naked’) molecular dye probes [1], which minimize phy- sical perturbation of the cell, but are subject to dye–cell chemical interference (e.g. protein binding, cell seques- tration and toxicity) [2], and pulled optical fiber chemical probes [3], which protect both the cell and the sensor from mutual chemical perturbation, but cause significant phy- sical disruption to the cell due to the probe insertion volume. By combining the advantages of both free mole- cular dyes and optical fiber probes, PEBBLEs come nearer to accomplishing the objective of a chemically and physically non-invasive sensor. PEBBLEs are nanosized sensors (20–100 nm), specif- ically designed for use in biological environments, which typically encapsulate an analyte-specific dye and a refer- ence dye within a biologically inert matrix [2,4]. Because of their small size, PEBBLEs are minimally physically disruptive to the cellular environment. Also, by encapsu- lating the dyes within an inert matrix, PEBBLEs create a sensing phase differentiated from the cell and, thereby, avoid chemical interference. PEBBLEs are a versatile platform that can be tailored to accommodate many dif- ferent sensing needs. PEBBLE sensors have been fabri- cated using four distinct matrices: polyacrylamide [2,4], polydecylmethacrylate (PDMA) [5–7], sol-gel [8], and organically modified silicates (ormosils) [9]. Hydrophilic dyes are best accommodated by polyacrylamide matrices, hydrophobic dyes by PDMA matrices, and amphiphilic dyes by either sol-gel or ormosil matrices. useful with hydrophilic, hydrophobic and amphiphilic dyes, respec- tively. Additional modifications may be made to the PEB- BLE platform to provide means for enhanced signal processing, targeted delivery, and so forth. In this review, we give an overview of the general principles governing PEBBLE design as well as different PEBBLE-sensing schemes, namely direct ion or small-molecule measure- ment and ion-correlation measurement, illustrating the discussion with a characteristic sensor for each scheme. We also outline typical results from in vitro cellular sensing. PEBBLE design Although many different PEBBLE platforms have been created, most follow a similar sensing scheme: analytes diffuse through the matrix and interact with the sensing dye, which elicits a change in the fluorescence detected from the PEBBLE. The sensors typically contain a reference dye; because the reference dye and sensing dye are both affected by variations in excitation intensity and other nonspecific interferences, the ratio between the two dyes eliminates distortion of the data by such phe- nomena. PEBBLE components are carefully chosen to utilize this ratiometric quality while optimizing the Current Opinion in Chemical Biology 2004, 8:540–546 www.sciencedirect.com