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Biomedical subjects

Robert M Corn

Publications and source records attributed to Robert M Corn.

At least 19 recordsLinked to original sources
PubMed2006

Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging.

RNA microarrays were created on chemically modified gold surfaces using a novel surface ligation methodology and employed in a series of surface plasmon resonance imaging (SPRI) measurements of DNA-RNA hybridization and RNA aptamer-protein binding. Various unmodified single-stranded RNA (ssRNA) oligonucleotides were ligated onto identical 5'-phosphate-terminated ssDNA microarray elements with a T4 RNA ligase surface reaction. A combination of ex situ polarization modulation FTIR measurements of the RNA monolayer and in situ SPRI measurements of DNA hybridization adsorption onto the surface were used to determine an ssRNA surface density of 4.0 x 10(12) molecules/cm2 and a surface ligation efficiency of 85 +/- 10%. The surface ligation methodology was then used to create a five-component RNA microarray of potential aptamers for the protein factor IXa (fIXa). The relative surface coverages of the different aptamers were determined through a novel enzymatic method that employed SPRI measurements of a surface RNase H hydrolysis reaction. SPRI measurements were then used to correctly identify the best aptamer to fIXa, which was previously determined from SELEX measurements. A Langmuir adsorption coefficient of 1.6 x 10(7) M(-1) was determined for fIXa adsorption to this aptamer. Single-base variations from this sequence were shown to completely destroy the aptamer-fIXa binding interaction.

Aptamers, Nucleotide↗
PubMed2006

Attomole microarray detection of microRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions.

Multiple microRNAs (miRNAs) are detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attomole levels.

Adenosine Monophosphate↗
PubMed2006

Surface plasmon resonance imaging measurements of antibody arrays for the multiplexed detection of low molecular weight protein biomarkers.

This paper describes a simple methodology for the creation of high-density multiplexed antibody arrays on gold surfaces that can be used to detect low molecular weight protein biomarkers with surface plasmon resonance imaging (SPRI). A one-step carbonyldiimidazole (CDI) surface reaction was utilized to attach antibodies onto alkanethiol-modified gold surfaces and characterized with polarization modulation FT-IR reflection absorption spectroscopy. The CDI chemistry was then employed to create an antibody microarray with array element sizes varying from 750 microm down to 200 microm. As a demonstration, a three-component antibody array was employed to detect two clinically important protein biomarkers, beta2-microglobulin (11.8 kDa) and cystatin C (13.4 kDa). SPRI measurements could simultaneously detect both of these small unlabeled proteins with no cross talk at solution concentrations from 300 nM down to 1 nM. In addition, the adsorption strengths of these biomarkers onto an antibody array were measured with SPRI and compared to those obtained from the kinetic analysis of single-channel angle shift SPR measurements.

Antibodies↗
PubMed2006

Creating advanced multifunctional biosensors with surface enzymatic transformations.

This paper summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.

DNA↗
PubMed2006

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions.

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.

DNA↗
PubMed2006

Surface plasmon resonance imaging measurements of protein interactions with biopolymer microarrays.

The surface-sensitive optical technique of surface plasmon resonance (SPR) imaging is an ideal method for the study of affinity binding interactions of unlabeled biological molecules in a multiplexed format. This approach has been widely applied to monitor DNA-DNA, DNA-RNA, peptide-protein, and protein-protein interactions as well as surface enzyme reactions. The success of SPR imaging measurements relies on the robust attachment of biomolecules in an array format. In this chapter, we introduce two different surface attachment chemistries that covalently immobilize DNA and peptides onto gold surfaces through the modification of self-assembled alkanethiol monolayers. Array fabrication approaches for the creation of individually addressable elements through the use of either gold dot patterns or polydimethylsiloxane (PDMS) microchannels are detailed. The utility of SPR imaging for the study of protein interactions is demonstrated with two biological systems: the binding of response regulator proteins, VanR and OmpR, onto a DNA array, and the interaction of S protein with an array of S-peptide variants. Furthermore, the application of real-time SPR imaging to the multiplexed determination of S-protein adsorption/desorption kinetics is described.

Bacterial Proteins↗
PubMed2005

Fabricating RNA microarrays with RNA-DNA surface ligation chemistry.

A novel surface attachment strategy that utilizes RNA-DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5'-phosphate-modified ssDNA attached to the surface and the 3'-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA-DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5'-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA-protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.

Adsorption↗
PubMed2005

Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy.

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.

Hydrolysis↗
PubMed2005

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays.

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.

DNA↗
PubMed2005

Long-range surface plasmon resonance imaging for bioaffinity sensors.

A novel bioaffinity sensor based on surface plasmon resonance (SPR) imaging measurements of a multiple-layered structure that supports the generation of long-range surface plasmons (LRSPs) at the water-metal interface is reported. LRSPs possess longer surface propagation lengths, higher electric field strengths, and sharper angular resonance curves than conventional surface plasmons. LRSPR imaging is a version of SPR imaging that requires a symmetric dielectric arrangement around the gold thin film. This arrangement is created using an SF10 prism/Cytop/gold/water multilayer film structure where Cytop is an amorphous fluoropolymer with a refractive index very close to that of water. LRSPR imaging experiments are performed at a fixed incident angle and lead to an enhanced response for the detection of surface binding interactions. As an example, the hybridization adsorption of a 16-mer single-stranded DNA (ssDNA) onto a two-component ssDNA array was monitored with LRSPR imaging. The ssDNA array was created using a new fabrication technology appropriate for the LRSPR multilayers.

Biosensing Techniques↗
PubMed2005

Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and Michaelis-Menten concepts.

Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] >> k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.

Adsorption↗
PubMed2004

Quantitative functional analysis of protein complexes on surfaces.

A major challenge in cell and molecular physiology research is to understand the mechanisms of biological processes in terms of the interactions, activities and regulation of the underlying proteins. Functional and mechanistic analyses of the large number of proteins that participate in the regulation of cellular processes will require new approaches and techniques for high throughput and multiplexed functional analyses of protein interactions, protein conformational dynamics and protein activity. In this review we focus on the development and application of proteomics and associated technologies for quantitative functional analysis of proteins and their complexes that include: (1) the application of surface plasmon resonance (SPR) imaging for multiplexed, label-free analyses of protein interactions, binding constants for biomolecular interactions and protein activities; and (2) high content analysis of protein motions within functional multiprotein complexes.

Animals↗
PubMed2004

Enzymatically amplified surface plasmon resonance imaging method using RNase H and RNA microarrays for the ultrasensitive detection of nucleic acids.

A novel surface enzymatic amplification method that utilizes RNA microarrays in conjunction with the enzyme RNase H is developed for the ultrasensitve detection and analysis of target DNA molecules. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from RNA-DNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance imaging, multiple DNA targets can be detected at a concentration of 10 fM on a single chip. In addition, this method is utilized for the sequence-specific detection of the TSPY gene in both purified and unpurified PCR products. Finally, in a series of kinetics measurements, the initial rate of hydrolysis is shown to depend directly on the surface concentration of DNA-RNA heteroduplexes.

Base Sequence↗
PubMed2004

Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays.

The kinetics of protein adsorption/desorption onto peptide microarrays was studied using real-time surface plasmon resonance (SPR) imaging. S protein binding interactions were examined using an array composed of five different peptides: N terminal and C terminal immobilized wild-type S peptide (S1 and S2), an alternate binding sequence derived by phage display (LB2), an NVOC-protected S peptide, and a FLAG peptide control sequence (F). Kinetic measurements of the S protein-S1 peptide interaction were analyzed to determine a desorption rate constant (k(d)) of 1.1 (+/-0.08) x 10(-2) s(-1), an adsorption rate constant (k(a)) of 1.9 (+/-0.05) x 10(5) M(-1) s(-1), and an equilibrium adsorption constant (K(Ads)) of 1.7 (+/-0.08) x 10(7) M(-1). SPR imaging equilibrium measurements of S protein to S1 peptide were performed to independently confirm the kinetically determined value of K(Ads). Rate constants for the S2 and LB2 peptides on the array were measured as follows: 1.6 (+/-0.04) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.07) x 10(-2) s(-1) (k(d)) for S2, 1.2 (+/-0.05) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.03) x 10(-2) s(-1) (k(d)) for LB2. In addition to S protein adsorption/desorption, real-time SPR imaging of peptide arrays was applied to study the surface enzymatic activities of the protease factor Xa. Enzymatic cleavage of the substrate peptide (P1) was shown to follow first-order kinetics and proceed at a rate 10 times faster than that of the mutant peptide (P2), with cleavage velocities of 5.6 (+/-0.3) x 10(-4) s(-1) for P1 and 5.7 (+/-0.3) x 10(-5) s(-1) for P2.

Adsorption↗
PubMed2004

Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays.

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.

DNA↗
PubMed2003

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions.

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.

Base Sequence↗
PubMed2003

Surface plasmon resonance imaging studies of protein-carbohydrate interactions.

Carbohydrate arrays fabricated on gold films were used to study carbohydrate-protein interactions with surface plasmon resonance (SPR) imaging. An immobilization scheme consisting of the formation of a surface disulfide bond was used to attach thiol-modified carbohydrates onto gold films and to fabricate carbohydrate arrays. The carbohydrate attachment steps were characterized using polarization modulation Fourier transform infrared reflection absorption spectroscopy; and poly(dimethylsiloxane) microchannels were used to immobilize probe compounds at discrete locations on a gold film. The binding of the carbohydrate-binding proteins concanavalin A (ConA) and jacalin to arrays composed of the monosaccharides mannose and galactose was monitored with SPR imaging. SPR imaging measurements were employed to accomplish the following: (i) construct adsorption isotherms for the interactions of ConA and jacalin to the carbohydrate surfaces, (ii) monitor protein binding to surfaces presenting different compositions of the immobilized carbohydrates, and (iii) measure the solution equilibrium dissociation constants for ConA and jacalin toward mannose and galactose, respectively. Adsorption coefficients (K(ADS)) of 2.2 +/- 0.8 x 10(7) M(-)(1) and 5.6 +/- 1.7 x 10(6) M(-)(1) were obtained for jacalin adsorbing to a galactose surface and ConA adsorbing to a mannose surface, respectively. The solution equilibrium dissociation (K(D)) constant for the interaction of jacalin and galactose was found to be 16 +/- 5 microM, and for ConA and mannose was found to be 200 +/- 50 microM.

Concanavalin A↗