Surface plasmon resonance investigations of human epidermal growth factor receptor 2.

This investigation utilizes surface plasmon resonance (SPR) spectroscopy to detect and quantify human epidermal growth factor receptor 2 (HER-2), an oncogene product that is over-expressed in some aggressive forms of breast cancer. Specifically, the HER-2 trans-membrane protein p185 and its extra cellular fragment p105 are analytes targeted in this work by using a gold-based biosensor slide on which an anti-HER-2 antibody has been immobilized by attachment to Protein G that is fixed to the gold film. A detection limit of > or =11 ng/mL for p185 resulted when trastuzumab was used as the anti-HER-2 antibody on the biosensor slide. Experiments with semi-purified p105 revealed that it binds weakly and reversibly to trastuzumab, therefore complicating its detection and quantification. Results of studies that reacted a 13-amino-acid peptide (PP13) from the HER-2 kinase domain with its specific antibody were critically different than p185 and p105 studies. Spectral analysis of the reflectivity at constant bulk buffer refractive index revealed a progressive negative SPR shift over time. A negative shift suggests that a loss of protein mass from the anti-PP13 antibody-Protein G biosensor is occurring. Several possibilities that may explain these negative SPR shifts are discussed.

Gas-phase detection of trinitrotoluene utilizing a solid-phase antibody immobilized on a gold film by means of surface plasmon resonance spectroscopy.

A multilayered biosensor was constructed and found to detect trinitrotoluene (TNT) in ppb concentrations in air both prior to and after detonation of TNT without use of a liquid phosphate buffered saline (PBS) superstrate. The biosensor surface was fabricated from a monoclonal antibody for TNT covalently bound to an 11,11'-dithio-bis(succinimidoylundecanoate) (DSU) self-assembled monolayer immobilized on a thin gold film bonded to a BK7 glass slide. The binding between the immobilized antibody and TNT antigen was detected using surface plasmon resonance spectroscopy (SPRS). Biosensor specificity for TNT was demonstrated with chemical homologues as well as against an unrelated explosive, RDX.

Picosecond absorption studies on the photodissociation of alpha- and beta-nitrosyl hemoglobin monomers.

Transient absorption studies of the pump-probe type were performed on the NO forms of the alpha- and beta-monomers of hemoglobin using a Nd3+ phosphate-glass laser. A second harmonic 531-nm, 8-ps fwhm pulse pumped the Q-band while a delayed continuum generated pulse was used to monitor pi pi* Soret absorption changes in the 410-453-nm region. Photodissociation of nitrosyl alpha- and beta-monomers was found to differ markedly from the tetramer in what we believe to be the formation of a five-coordinate HbNO (with proximal imidazole detached) photoproduct within the first 50 ps after photon absorption.

Photodissociation of CO and O2 from alpha and beta hemoglobin chains studied by using picosecond absorption spectroscopy.

The picosecond photodissociation of the CO and O2 forms of alpha and beta chains of hemoglobin were studied by following pi pi Soret absorption changes using a Nd3+ phosphate-glass laser, 531-nm pump pulse, 8 ps full width half maximum, and a pump-probe double-beam absorption apparatus. Three intermediates were observed within the first 50 ps after photon absorption. The most notable differences between the two monomers are the extent and rate of geminate recombination with the two ligands. We attribute this result to differences between the tertiary protein structure of the alpha and beta forms of Hb, both distal and proximal.

Kinetics of carboxymyoglobin and oxymyoglobin studied by picosecond spectroscopy.

Picosecond studies of carboxymyoglobin (MbCO) and oxymyoglobin (MbO2) reveal that excitation at 530 nm induces photodissociation at less than 8 ps. The kinetic and structural changes were monitored by following absorbance changes at selected wave-lengths in the Soret (B) band and in the Q band. Within the 10 ps-0.45 ns period of time over which our experiments were conducted, the absorbance changes in the Soret and Q bands for MbCO and MbO2 correspond to the conventional long-term, steady-state deoxymyoglobin difference spectra (Mb-MbCO and Mb-MbO2), as determined by comparison of isosbestic, maximum, and minimum points. In addition, MbCO exhibits a decay to a steady state in the Soret band (monitored at 440 nm). The onset of the decay immediately follows photodissociation and has a rate of (8 +/- 3) X 10(9) s-1 (tau = 125 +/- 50 ps). During the 10 ps-0.45 ns observation window, relaxation is not seen for MbO2 in the Soret band, nor is relaxation observed in the Q band for either MbCO or MbO2. We conclude from these results that the steady state that we observed for MbCO and MbO2 is most likely the stable form of deoxymyoglobin, and the relaxational differences between MbCO and MbO2 observed in the Soret band indicate that the electronic destabilization after ligand detachment is very different for these molecules. We believe that these relaxational differences may be related to differences in tertiary structural changes, or due to the fact that the MbCO (S = 0) molecule passes through an intermediate spin Mb (S = 1) state before relaxing the the Mb (S = 2) state.

Picosecond photodissociation and subsequent recombination processes in carbon monoxide hemoglobin.

Excitation of HbCO by a single 6-psec 530-nm pulse results in photodissociation with a first-order constant of 0.89 X 10(11) sec-1. The kinetics of photodissociation, monitored by following absorbance changes in the Soret band at 440 nm, are interpreted as corresponding to predissociation followed by a corssing into a dissociative state. Subsequent recombination of CO with the porphyrin system and protein structural transformations were monitored by use of a continuous He-Cd laser beam spatially coincident with the photolysis and Soret interrogation beams at the sample. We find that the latter events take place in three distinct time regions, depending on exciation pulse energy and repetition rate. Exictation of HbCO with a single pulse (0.8--5 mJ) results in a relaxation to the ground state with an associative first-order constant of 5 X 10(3) sec-1. With a 100-pulse train (approximately 7.5 mJ), a new decay grows with a rate constant of 63 sec-1. For a pulse-train energy of 12 mJ or higher, a delay occurs at the onset of the second (slower) recombination.