Fluorescence quenching competitive immunoassay in micro droplets Jun Feng a , Guomin Shan b , Bruce D. Hammock b , Ian M. Kennedy a, * a Department of Mechanical and Aeronautical Engineering, University of California, Davis, CA 95616, USA b Department of Entomology and Cancer Research Center, University of California, Davis, CA 95616, USA Received 4 February 2002; received in revised form 20 September 2002; accepted 25 September 2002 Abstract A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-mm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 /512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen  /antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen  /antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence; Micro droplet; Quenching; Immunoassay; Esfenvalerate 1. Introduction Laser induced fluorescence technology, combined with optical microscope systems and sensitive charged- coupled device (CCD) cameras, has become an impor- tant tool in biological research (Meixner and Kneppe, 1998; Nie and Zare, 1997; Peck et al., 1997; Powell and Tempst, 2001; Ronai et al., 2001; Tuma et al., 1999). However, background interference is still a major challenge to the more general application of this method, particularly in environmental sciences where the matrix that contains the compound of interest may quench a fluorescence signal, may autofluoresce, or may adversely influence the assay in other ways. In addition to high-performance optical filters, reduc- tion in the illuminated sample volume is an effective way to overcome background interference in laser induced fluorescence detection. Several schemes have been explored to achieve this goal. Fluorescence detection in hydrodynamically focussed liquid streams has shown significant advantages in reducing background interfer- ence to the level at which single molecules can be detected (Nie et al., 1995; Shera et al., 1990; Soper et al., 1992). Capillary electrophoresis has also aided in the detection of single molecules (Castro and Shera, 1995; Chen and Dovichi, 1996). Other analysis formats can be useful in extending the limits of detection. Micro droplets (droplets +/100 mm) can be used to define the interaction volume of laser and analyte very precisely. The sample volumes are typically in the picoliter range. Electrodynamic levitation of picoliter-sized droplets (Barnes et al., 1993; Kung et al., 1998; Lermer et al., 1997) has been used to measure a single droplet for an extended period of time in order to improve the signal to noise ratio (SNR). In addition, micro droplets can remove the possibility of non-specific adsorption on the walls of cuvettes that are used to hold and analyze samples (Welter and Neidhart, 1997). The * Corresponding author. Tel.:  /1-530-752-2796; fax:  /1-530-210- 8220 E-mail address: imkennedy@ucdavis.edu (I.M. Kennedy). Biosensors and Bioelectronics 18 (2003) 1055 /1063 www.elsevier.com/locate/bios 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0956-5663(02)00218-X