A combination of scanning and imaging surface plasmon resonance (SPR) experiments is used to characterize DNA hybridization adsorption at gold surfaces and the subsequent immobilization of streptavidin. Single-stranded oligonucleotides are immobilized at gold surfaces, and the hybridization of biotinylated complements from solution is monitored with SPR. The subsequent attachment of streptavidin to the biotinylated complements provides a method of enhancing the SPR imaging signal produced as a result of the hybridization and leads to a 4-fold improvement in the hybridization detection limit of the SPR imaging apparatus. In situ scanning SPR experiments are used to measure a 60 ± 20% hybridization efficiency between immobilized single-stranded DNA and biotinylated complements. From the information provided by both the in situ imaging and scanning SPR experiments, an absolute surface coverage of immobilized single-stranded DNA is estimated to be ∼3 × 1012 molecules/cm2. The SPR signal resulting from hybridization onto immobilized probes is further amplified by the formation of streptavidin/DNA multilayers which grow by a combination of DNA hybridization and biotin−streptavidin binding. DNA/DNA multilayers without streptavidin are used as an additional method of amplifying the SPR signal.

A new method for constructing oligonucleotide arrays on gold surfaces has been developed, and these arrays have been used in DNA hybridization experiments with in situ surface plasmon resonance (SPR) imaging detection. The detection technique was able to differentiate between single- and double-stranded DNA regions on the gold surface. The hybridization of both oligonucleotides and PCR-amplified DNA fragments was detectable, with the latter exhibiting slower hybridization kinetics. Temperature control of the in situ SPR cell was used to discriminate between perfectly matched duplexes and single-base-mismatched duplexes. The SPR detection technique requires no label on the DNA, but fluorescently labeled targets were also tested and detected by fluorescence imaging as an independent verification of the hybridization behavior of these DNA arrays. The in situ SPR imaging method for detection of DNA hybridization is expected to complement other existing methods for study of DNA interactions and might find future uses in mutation screening assays and DNA resequencing.

Monolayers of the polypeptide pofy(L-lysme) (PL) are used to control the specific adsorption of proteins onto gold surfaces. A PL monolayer modified with biotin is electrostatically adsorbed onto a vapor-deposited gold film that has been coated with a self-assembled monolayer of the alkanethiol 11-mercaptoundecanoic acid (MUA). The immobilized biotin moieties act as specific adsorption sites for the protein avidin. Adsorption of the biopolymers onto the gold surface is monitored with a combination of surface plasmon resonance (SPR) and fluorescence measurements. By varying the percent biotinylation of the lysine residues on the PL prior to deposition, the surface coverage of avidin can be controlled to create either full or partial monolayers. The thickness of a full monolayer of avidin is 41 A, as determined by the SPR measurements. At high surface coverages of avidin, an excess of biotin sites is required to overcome steric hindrance. The PL monolayer and any adsorbed avidin can be easily rinsed from the surface with a low or high pH solution. This removal allows for quantitation of the adsorbed molecules by fluorescence measurements in solution rather than on the gold surface. In this manner, fluorescein-labeled PL and avidin are used to determine absolute surface coverages of 4 x 1014 lysine residues cm-2 for the PL monolayer and 3 x 1012 avidin molecules cm-2 for the full avidin monolayer. SPR imaging experiments are employed to verify that UV photopatteming of the MUA/PL bilayers can be used to spatially direct the adsorption of avidin onto the gold surface. The polyfysine attachment methodology will be beneficial in the fabrication of adsorption biosensors.The fabrication of bioanalytical devices such as adsorption biosensors, enzyme-coated electrodes, and affinity chromatography columns often requires the attachment of protein molecules onto a solid surface.1-3 In this attachment step, it is frequently necessary to control the specific adsorption of a particular protein and to prevent the nonspecific adsorption of other biological

A combination of in situ and ex situ surface plasmon resonance (SPR) imaging experiments is used to characterize the differential electrostatic adsorption of proteins and synthetic polypeptides onto photopatterned monolayers at gold surfaces. The nonspecific electrostatic adsorption of proteins onto negatively charged self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) is found to depend on the protein pI, solution ionic strength, and solution pH. The pH dependence of the electrostatic adsorption of the protein avidin onto a MUA SAM indicates that a full monolayer adsorbs at a solution pH greater than 5.0, and an "effective pK(a)" of 3.6 is determined for the avidin adsorption. This effective pK(a) is a combination of the pK(a) of the MUA monolayer and the ion pairing adsorption coefficient for the avidin. Additional SPR imaging experiments show that the electrostatic adsorption of the synthetic polypeptide poly-l-lysine (PL) onto a MUA SAM varies with molecular weight, forming a full PL monolayer for polypeptides with more than 67 lysine residues.

No abstract