Single-particle based counting is not only a reasonable approach to achieve high sensitivity without the aid of target amplification, but also impart the method with unbiased quantification of multiple /ytes. Based on dark-field microscopic imaging of gold nanoparticles and fluorescence microscopic imaging of fluorescent nanoparticles/microspheres, we have developed microscopic imaging based nanoparticle counting methods for single and multiple /ytes in one test volume. Using colocalized fluorescence nanoparticle counting, we can achieve the six DNAs/miRNAs with less than 10 fM limit of detection in one test sample, and Endonucleases/DNA Methyltransferases in one test sample. Based on quantum dot doped nanoparticle counting, we can achieve simultaneous detection of proteins and miRNAs in one test sample. These methods show the potential of applications in biological samples. We will continue to develop single particle counting based method for sensitive and multiplexed assays.
