This review with 60 references describes a unique path to miniaturisation, that is, the use of acoustic levitation in analytical and bioanalytical chemistry applications. Levitation of small volumes of sample by means of a levitation technique can be used as a way to avoid solid walls around the sample, thus circumventing the main problem of miniaturisation, the unfavourable surface-to-volume ratio. Different techniques for sample levitation have been developed and improved. Of the levitation techniques described, acoustic or ultrasonic levitation fulfils all requirements for analytical chemistry applications. This technique has previously been used to study properties of molten materials and the equilibrium shape()and stability of liquid drops. Temperature and mass transfer in levitated drops have also been described, as have crystallisation and microgravity applications. The airborne analytical system described here is equipped with different and exchangeable remote detection systems. The levitated drops are normally in the 100 nL-2 microL volume range and additions to the levitated drop can be made in the pL-volume range. The use of levitated drops in analytical and bioanalytical chemistry offers several benefits. Several remote detection systems are compatible with acoustic levitation, including fluorescence imaging detection, right angle light scattering, Raman spectroscopy, and X-ray diffraction. Applications include liquid/liquid extractions, solvent exchange, analyte enrichment, single-cell analysis, cell-cell communication studies, precipitation screening of proteins to establish nucleation conditions, and crystallisation of proteins and pharmaceuticals.

A miniaturized analysis system for the study of living cells and biochemical reactions in microdroplets was developed. The technique utilizes an in-house-developed piezoelectric flow-through droplet dispenser for precise reagent supply and an ultrasonic levitator for contactless sample handling. A few-cell study was performed with living primary adipocytes. Droplets (500 nL) containing 3-15 individual cells were acoustically levitated. The addition of beta-adrenergic agonists into the levitated droplet using the droplet dispenser stimulated adipocyte lipolysis, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer. The addition of insulin antagonized lipolysis and hence also the decrease in pH. The changes in pH, i.e., the cell response in the droplet, were followed using a pH-dependent fluorophore continuously monitored by fluorescence imaging detection. An image analysis computer program was employed to calculate the droplet intensities. To counteract droplet evaporation, found to affect the fluorescence intensities, a separate dispenser was used to continually add water, thus keeping the droplet volume constant.

Laser techniques were applied to an acoustically levitated droplet for remote investigation of the diameter, species concentration and temperature of the suspended droplet. To this end, the third and the fourth harmonic of a Nd:YAG laser were used for investigation of elastic, fluorescence and phosphorescence signals from the droplet. The droplet was seeded with thermographic phosphors and acetone for the phosphorescence and fluorescence measurements, respectively. The techniques were applied simultaneously using an imaging stereoscope. The imaging device allowed for an identical visualization of incoming signal through separate optical filters. Temperature measurements in droplets is important in the study of e.g. exothermic chemical reactions, spray processes, combustion, and in bioanalytical applications where the biological material is temperature sensitive or dependent on optimal temperature for function. Results from these investigations showed that temperature measurements in acoustically levitated droplets using laser-induced phosphorescence are feasible. The results also show the potential of simultaneous laser based measurements on levitated droplets. Diameter variation (surface area), mixture concentration and temperature were measured simultaneously.

The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-microL volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the viscous alcohol 2-methyl 2,4-pentanediol. From a digitized picture of the levitated drop volume, calculations are performed giving the concentrations of all components in the drop at any time during a "precipitation experiment". Supersaturation is the prerequisite for crystal nucleation, and protein precipitation indicates high supersaturation. A light source illuminates the levitated drop, and protein precipitation is monitored using right-angle light scattering. On the basis of these intensity measurements and the volume determination, precipitation diagrams for each crystallizing agent are constructed that give the protein/crystallizing agent concentration boundaries between the minimum and the maximum detectable protein precipitation. Guided by the concentration values obtained from such plots, when approaching the supersaturation region, separate crystallization drops are mixed and allowed to equilibrate under paraffin oil. At conditions in which microcrystals can be observed, the nucleation tendency of the macromolecule is confirmed. Optimization of crystallization conditions can then follow. Proteins tested include alcohol dehydrogenase and D-serine dehydratase. Alcohol dehydrogenase, known to crystallize easily, was used to evaluate whether the ultrasonic field inhibits nucleation. Details are given for the screening procedure of D-serine dehydratase, an enzyme earlier found to be difficult to crystallize reproducibly. The time and material-saving qualities of this method are emphasized, since a range of conditions can quickly be screened using small amounts of protein to roughly determine solubility characteristics of a protein before crystallization trials are initiated.

In this paper, the use of airborne chemistry (acoustically levitated drops) in combination with Raman spectroscopy is explored. We report herein the first Raman studies of crystallization processes in levitated drops and the first demonstration of surface-enhanced Raman scattering (SERS) detection in this medium. Crystallization studies on the model compounds benzamide and indomethacin resulted in the formation of two crystal modifications for each compound, suggesting that this methodology may be useful for investigation of polymorphs. SERS detection resulted in a signal enhancement of 27 000 for benzoic acid and 11 000 for rhodamine 6-G. The preliminary results presented here clearly indicate that several important applications of the combination between Raman spectroscopy and acoustic drop levitation can be expected in the future.