The potentiometric protein immunoassay exhibits a logClinear response ranging from 0.15 to 4.0 pmol of IgG, with a detection limit of 10 fmol in 150-L sample wells. Nanoparticle-based electrochemical protein immunoassays typically employ stripping voltammetric techniques as the readout principle.1C6 Upon completion of the protein binding event with a secondary antibody labeled with gold nanoparticles or semiconductor quantum dots, the tracers are detected electrochemically. labeled with gold nanoparticles or semiconductor quantum dots, the tracers HG6-64-1 are detected electrochemically. This may be achieved after an oxidation/dissolution step with reagents such as nitric acid7 or directly with solid-state chrono-potentiometry.8 Alternatively, enzyme labels, known as highly effective biological amplifiers, can be used, for example, in a magnetic collection on interdigitated electrodes.9 Recently, carbon nanotubes decorated with multiple enzymes were also used as labels for an even greater chemical amplification of the immunobinding event.10 Stripping voltammetric techniques exhibit extremely low lower detection limits in terms of concentration. The measurement of trace level concentrations in microliter or submicroliter volumes has been achieved with nanoband electrodes. In 500-nL HG6-64-1 sample droplets, the lowest detected concentrations were 0.5 ppb lead with high-amplitude square wave voltammetry,11 while the injection of 1 1.4-L HG6-64-1 samples of tear fluid made it possible to measure 6.5 ppb cadmium with anodic stripping voltammetry,12 both with mercury film electrodes. In these two examples, the detected total amounts of material were on the order of 2.5 10?13 and 6.5 10?13 g of lead and cadmium, respectively. More recent efforts aimed at further reducing the detection volume down to 1 nL, but the lowest detected amount of material was somewhat inferior at 7.5 10?12 g.13 Potentiometry with ion-selective electrodes (ISEs) is attractive for trace level analysis in confined samples. There exists a direct relationship between sample activity and observed electromotive force, independent of the volume of the sample or electrode surface. Indeed, ion-selective microelectrodes have been used for the detection of millimolar concentrations in single cells having volumes on the order of 1 1 pL.14 In recent years, polymer HG6-64-1 membrane potentiometric sensors have reached ultratrace level detection limits on the order of nanomolar or lower concentrations.15,16 This was achieved by understanding and minimizing undesired zero current ion fluxes from the membrane toward the sample, which have traditionally tainted the electrode response. With micropipet-based ISEs, it was recently found that the lower detection limit can be approximately maintained in confined sample volumes.17 The detection of 10?10 M Ag+, Pb2+, and Ca2+ was demonstrated in ultrasmall volumes of 3 L, resulting in a detectability of 300 amol.17 Potentiometric immunoassays have been explored by only a limited number of researchers. Rechnitz and co-workers introduced gas-sensing probes in conjunction with NH3-producing deamination enzymes such as asparaginase as labels18 or with the CO2 production from -ketoadipic acid by chloroperoxidase enzyme-labeled IgG antibody.19 Others used an I?-selective electrode to monitor the extent of oxidation of I? by H2O2 produced by a peroxidase label.20 More recently, Koncki and coworkers used a F?-selective electrode for monitoring the release of F? from the substrate monofluorophosphate, catalyzed by the alkaline phosphatase label.21 Meyerhoffs group explored polycation-selective electrodes to monitor immunoreactions in a competitive homogeneous assay format.22 The recent progress with ISEs IL-2 antibody described above forms the basis for the application of potentiometric sensors in ultrasensitive bioanalysis using chemical amplification steps. Recently, we reported on the detection of proteins in a potentiometric immunoassay with gold nanoparticle labels that were chemically plated with silver.23 The chemically dissolved silver ions were subsequently detected by potentiometry. Here, we explore the detection of Cd2+ with a Cd2+-selective micropipet electrode in a more convenient format, utilizing commercially available CdSe semiconductor quantum dots as labels for protein immunoassays without any further chemical enhancement step, and using microtiter plates as a readout platform. The detection limit is lower by ~3 orders of magnitude than reported previously.23 EXPERIMENTAL SECTION Reagents, Membranes, and Electrodes See Supporting Information. Emf Measurements Potential measurements were performed with a 24-bit emf measuring interface (EMF-16, Lawson Labs, Malvern, PA) with 1 reference and 16 measuring channels in a Faraday cage. Measurements in conventional 100-mL samples were performed at ambient temperature using a commercial reference electrode (Metrohm, No. 6.0729.100, Metrohm, CH-9101 Herisau) with a 1 M NH4NO3 electrolyte..