© 2009 Macmillan Publishers Limited. All rights reserved. Polymer nanofibre junctions of attolitre volume serve as zeptomole-scale chemical reactors Pavel Anzenbacher, Jr * & Manuel A. Palacios Methods allowing chemical reactions to be carried out on ultra-small scales in a controllable fashion are potentially important for a number of disciplines, including molecular electronics, photonics and molecular biology, and may provide fundamental insight into chemistry in confined spaces. Ultra-small-scale reactions also circumvent potential problems associated with reagent and product toxicity, and reduce energy consumption and waste generation. Here, we report a technique for performing chemical reactions on a zeptomole (10 221 mol) scale. We show that electrospun polymer nanofibres with a diameter of 100–300 nm can be loaded with reactants, and that the junctions formed between crossed nanofibres can function as attolitre-volume reactors. Exposure to heat or solvent vapours fuses the fibres and initiates the reaction. The reaction products can be analysed directly within the nanofibre junctions by fluorescence measurements and mass spectrometry, and solvent extraction of multiple reactors allows product identification by common micromethods such as high-performance liquid chromatography–mass spectrometry. F rom the perspectives of both fundamental and applied chem- istry, the ability to carry out chemical reactions at a sub-atto- mole scale is highly desirable. If performed in a reliable and controlled manner, such methods would benefit numerous research and development efforts by providing data on relative reactivity alongside high-throughput liquid chromatography–mass spec- trometry (LC-MS) analyses, while using small samples for prelimi- nary analyses where reagent/product stability is a concern 1,2 . Other desirable features of these methods include circumvention of poten- tial problems associated with reagent and/or product toxicity and reduction of the amounts of reagents used, and energy consumption and waste during initial reaction testing. On a more fundamental level, methods for detection of chemical species reaching detection limits as small as individual molecules are available 3,4 . These include methods based on metal nanoparticles 5 or semiconductor quantum dots 6 , as well as methods using gas chromatography 7 or capillary electrophoresis 8 that allow the detection of ultra-low concentrations of individual components, which may be analysed spectroscopi- cally 9 or by mass spectrometry 10 . Whereas analytical techniques have made strides towards sub- attomole detection limits, reactors capable of the preparation of chemical species on an ultra-small scale are not readily available, are expensive or do not allow effective control of chemical reac- tions 11 . This seems to be largely due to the complex methods of microreactor fabrication 11,12 . In the case of sub-nanolitre-volume reactors, two main approaches have emerged: a top-down method of reactor fabrication using microfluidics 13 and ‘lab-on-a-chip’ technologies 14,15 . The use of nanodroplets transported through microfluidic chan- nels in a synchronized fashion allows reagent-carrying droplets to react in a controlled manner while eliminating dispersion 16,17 , accel- erating mixing 18 and providing control over the chemistry 19 . This technique also allows screening of reactions, including nanoparticle synthesis 20 , protein crystallization 21 , DNA assays 22 , organic synth- eses 23 and combinatorial screening 24 . The bottom-up approach is best illustrated by self-assembled molecular-scale reactors 25 . These can use inorganic materials such as zeolites 26 and other mesoporous materials 27 . An alternative approach based on organic materials uses molecular reagent-sized cavities in self-assembled capsules 28 , imprinted polymers 29 or supra- molecular containers 30 . Some molecular-scale reactors exhibit cata- lytic activity, and in several cases impressive efficiencies have been observed 31,32 . In spite of the indisputable achievements, most of these techniques seem to be far from easy to implement, and the attendant costs of synthesis and device preparation remain an obstacle. We present here an easy-to-perform and versatile method enabling chemical reactions in femto- to attolitre volumes on a scale down to 1,000 molecules, that is, the zeptomole (1 zmol ¼ 10 221 mol) scale. In theory, this method allows reactions to be carried out that potentially scale down to two single molecules. The method is based on the deposition of a rectangular grid of two nanofibre types, each nanofibre being doped with a different reagent (Fig. 1a). Heat or solvent vapour-welding of the softened polymer (for example poly- urethane Tecoflex) nanofibres results in mixing of the contents of the fibres at the intersection, thereby establishing a mixed junction. In the example illustrated in Fig. 1b–d, polymer electrospinning 33 was used to fabricate rectangular nanofibre grids 34,35 , and the fibre fusion was performed by heating or simple exposure to solvent vapours. Solvent-vapour-mediated coalescence (similar to the ‘welding’ of polymer nanofibres demonstrated earlier by Sotzing and co-workers 36 and to careful heat-welding of overlaid fibres in the form of nanofibre mats) does not result in collapse of the fibrous structure, and thus allows multiple junctions to be established in a small volume. Figure 2 shows an example of a reaction carried out in a nano- fibre junction. Neither of the starting materials, dansyl chloride (1-dimethylaminonaphthalene-5-sulfonyl chloride, 1) and triethy- lenetetramine (2), is fluorescent. The product, a dansylamide (3), is brightly fluorescent 37 , which allows easy visualization of the reac- tion carried out in the attoreactor. After the nanofibre junction fusion, a combination of surface profilometry and scanning electron microscope (SEM) measurements indicates that the junctions are 90 nm high and 200 nm wide. The average volumes of the Center for Photochemical Sciences and Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, USA. *e-mail: pavel@bgsu.edu ARTICLES CORRECTED ONLINE: 16 AND 27 MARCH 2009 PUBLISHED ONLINE: 8 MARCH 2009 | DOI: 10.1038/NCHEM.125 NATURE CHEMISTRY | VOL 1 | APRIL 2009 | www.nature.com/naturechemistry 80