Molecular-Counting-Free and Electrochemiluminescent Single- Molecule Immunoassay with Dual-Stabilizers-Capped CdSe Nanocrystals as Labels Xin Zhang, † Bin Zhang, † Wujian Miao, ‡ and Guizheng Zou* ,† † School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China ‡ Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States * S Supporting Information ABSTRACT: Biorelated single-molecule detection (SMD) has been achieved typically by imaging the redox fluorescent labels and then determining each label one by one. Herein, we demonstrated that the capping agents (i.e., mercaptopropionic acid and sodium hexametaphosphate) can facilitate the electrochemical involved hole (or electron) injecting process and improve the stability of the dual-stabilizers-capped CdSe nanocrystals (NCs), so that the CdSe NCs could be electrochemically and repeatedly inspired to excited states by giving off electrochemiluminescence (ECL) in a cyclic pattern. With the CdSe NCs as ECL label and carcinoembryonic antigen (CEA) as target molecule, a convenient single-molecule immunoassay was proposed by simply detecting the ECL intensity of the dual-stabilizers-capped CdSe NCs in a sandwich-typed immune complex. The limit of detection is 0.10 fg/mL at S/N = 3, which corresponds to about 6-8 CEA molecules in 20 μL of serum sample. Importantly, the ECL spectra of both CdSe NCs and its conjugate with probe antigen in the immune complex were almost identical to the photoluminescence spectrum of bare CdSe NCs, indicating that all emissions were originated from the same excited species. The molecular-counting-free and ECL-based SMD might be a promising alternative to the fluorescent SMD. T he detection and identification of single molecules (i.e., single-molecule detection (SMD)) means to determine all the target molecules in a given volume, 1 which represents the ultimate limit in chemical analysis and medical diagnostics, 2 and can enable both fundamental studies and new analytical applications. 3,4 As the signal produced by a single molecule is extremely weak, how to minimize the background signals, optimize the overall signal efficiency, and amplify the signal play crucial roles in SMD. 5-8 Because a redox fluorescent molecule or nanocrystal (NC) can emit many photons and result in intrinsically amplified signal before it is photobleached, biorelated SMD were usually achieved with redox fluorescent molecules or NCs as labels to image the target and then figure them out one by one in a “molecular-counting” way. 9-11 On the contrary, because only a microscopic amount of charge was involved in the electrochemical reaction per molecule, 5 even with the most efficient redox molecule, the electrochemical response of a single molecule is still near the limits of detectability (about 10 -15 A), 3,12,13 and the electrochemical SMD was strictly limited to detecting simple redox molecules with unconventional electrochemical cells and measure- ments. 5,6,14,15 Only a few biorelated electrochemical SMD techniques have been proposed to determine the targets by counting the magnetic nanobeads, which were proportional to the targets at a mole ratio around 1:1, with the combined nanobead amplification and enzyme amplification techniques, 16 which is time-consuming, laborious, and external-signal- amplifying-dependent. New approaches for biorelated SMD are still strongly anticipated. Electrochemiluminescence (ECL) is an electro- chemically triggered optical radiation produced by the energy relaxation of excited species, 17 which is superior to fluorescence in terms of sensitivity and signal-to-noise ratio due to absence of background from unselective photoexcitation. 18 With enlarged labeling ratios (i.e., an external-signal-amplifying strategy), the limit of detection (LOD) for ECL has been improved to a femtomolar level by now. 19-21 Actually, the fluorescent SMD labels (i.e., NCs) have also demonstrated promising ECL behavior from ensembles 22-24 to individual nanoparticles. 25 However, because minimal research concern- ing the redox ECL of NCs has been carried out for intrinsically signal-amplifying strategy and because biorelated ECL analyses were usually performed with various external-signal-amplifying strategy, 26 their performance was far from the single-molecule level. Recently, with mercaptopropionic acid (MPA) and sodium hexametaphosphate (HMP) as capping agents, we developed a Received: March 11, 2016 Accepted: April 27, 2016 Article pubs.acs.org/ac © XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b00967 Anal. Chem. XXXX, XXX, XXX-XXX