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The interaction of the antitumor drug cisplatin with insulin was studied using a top-down mass spectrometric approach. In vitro incubations were prepared under acidic and physiological conditions at different insulin/cisplatin molar ratios for different incubation times. Size exclusion chromatography--inductively coupled plasma mass spectrometry (SEC--ICPMS) analysis enabled the specific detection of platinum containing species attributed to the binding of the drug to the protein. Further analysis through matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoelectrospray ionization mass spectrometry using a linear ion trap (nESI-LIT-MS) allowed the identification of platinated mono-, di-, and even triadducts in the incubations. Platinum binding sites were identified by [CID-MS.sup.n] as B chain N-terminus, His5, and probably His10 residues, which turned out to be the same, regardless of the incubation conditions. Evidence on the binding of Pt to B chain Cys7 was also observed. Working with the LIT zoom scan mode provides enough resolution to discern the isotopic pattern for both precursor and fragment ions, allowing the differentiation of platinum-containing ions. The elucidation of platinum binding sites in a native protein through a top-down approach has been performed for the first time with this type of instrument.

We present a fractional sensitivity analysis of a competitive microfluidic heterogeneous immunoassay for a small molecule analyte. A simple two-dimensional finite element model is used to determine the fractional sensitivity of the assay signal with respect to analyte concentration, flow rate, initial surface density of binding sites, and antibody concentration. The fractional sensitivity analysis can be used to identify (1) the system parameters for which it is most crucial to control or quantify the variability between assays and (2) operating ranges for these parameters that improve assay sensitivity (within the constraints of the experimental system). Experimental assay results for the drug phenytoin, obtained using surface plasmon resonance imaging, are shown to be consistent with the predictions of the model.