Interferometry and Fluorescence Detection for Simultaneous Analysis of Labeled and Unlabeled Nanoparticles in Solution Stefan Wennmalm* and Jerker Widengren Royal Institute of Technology, Albanova University Center, Department of Applied Physics, Experimental Biomolecular Physics, 106 91 Stockholm, Sweden * S Supporting Information ABSTRACT: A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward- scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K d from a single binding measurement. T he two most popular techniques for label-free analysis of particles in solution are dynamic light scattering 1 (DLS) and laser diffraction spectroscopy (LDS). 2 While DLS derives particle size from the diffusion coefficient, LDS measures the particles’ projected cross section. However, neither of these techniques estimates the concentration of particles, and they cannot easily be combined with fluorescence techniques. In the past few years, interferometric techniques for analysis of single metal and polymer nanoparticles (NPs) and viruses have gained much interest. They offer high-sensitivity detection of unlabeled nanosized objects 3,4 but also allow metal NPs to be used as an alternative label that is free from fluorescence bleaching, blinking, and saturation. 5-8 Photothermal correla- tion spectroscopy 9 (PCS) and photothermal absorption correlation spectroscopy 10 (PhACS) were recently demon- strated as interferometric techniques for solution analysis of gold NPs as an alternative specific label. Here scattering interference correlation spectroscopy (SICS) is introduced as a label-free technique in which fluctuations are likely caused by interference between the phase-shifted forward scattering from NPs and the transmitted laser light (reference beam), as in PCS and PhACS (Figure 1A). Autocorrelation of the forward-scattered and transmitted light yields information about not only the NPs’ hydrodynamic radius but also their effective cross section and concentration. Furthermore, we demonstrate how the technique easily can be combined with fluorescence correlation spectroscopy (FCS) to allow simulta- neous analysis of labeled and unlabeled NPs. The autocorrelation function (ACF) amplitude is given by δ − = ⟨ ⟩ ⟨⟩ G I I (0) 1 (0) 2 2 (1) where I is the detected intensity, δI(t) is the deviation from the mean intensity at time t, and brackets denote the mean value. The signal caused by a single unlabeled NP is σ p P tot /A dv = P tot A q , where σ p is an effective cross section, P tot is the applied laser power, A dv is the area of the laser focus, and A q is the normalized effective cross section (A q = σ p /A dv ). With a mean number of particles N in the detection volume, the fluctuation Received: August 18, 2012 Published: November 16, 2012 Figure 1. (A) Experimental setup for SICS and its combination with FCS. (B) Intensity trace and (C) histogram of a measurement on 93 nm NPs (bin time = 8 ms, diffusion time τ D =8-9 ms, N = 0.18). (D) Intensity trace and (E) histogram of a measurement on pure buffer solution (bin time = 8 ms). Measurement times were 120 s. Communication pubs.acs.org/JACS © 2012 American Chemical Society 19516 dx.doi.org/10.1021/ja308213q | J. Am. Chem. Soc. 2012, 134, 19516-19519