Eric Schopf

Protein nanopatterns by oxime bond formation.

Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies provide site-specific attachment of the biomolecules. In this study, oxime chemistry was exploited to conjugate proteins onto nanometer-sized features. Poly(Boc-aminooxy tetra(ethylene glycol) methacrylate) was synthesized by free radical polymerization. The polymer was patterned onto silicon wafers using an electron beam writer. Trifluoroacetic acid removal of the Boc groups provided the desired aminooxy functionality. In this manner, patterns of concentric squares and contiguous bowtie shapes were fabricated with 150-170-nm wide features. Ubiquitin modified at the N-terminus with an α-ketoamide group and N(ε)-levulinyl lysine-modified bovine serum albumin were subsequently conjugated to the polymer nanopatterns. Protein immobilization was confirmed by fluorescence microscopy. Control studies on protected surfaces and using proteins presaturated with O-methoxyamine indicated that attachment occurred via oxime bond formation.

Dual Click reactions to micropattern proteins

In the study described in this report orthogonal Click reactions were utilized to immobilize two different proteins on surfaces side-by-side and in multilayer constructs. Alkyne- and azide-functionalized poly(ethylene glycol) hydrogel features were fabricated. Copper-catalyzed Huisgen 1,3 dipolar cycloaddition and oxime chemistry were employed to conjugate an azide-functionalized ubiquitin and oxoamide-modified myoglobin, respectively. Multicomponent patterning was verified by fluorescence imaging.

Attomole DNA detection assay via rolling circle amplification and single molecule detection

A bead-based assay was developed for highly sensitive single molecule DNA detection. Rolling circle amplification (RCA), an isothermal amplification technique that creates tandem repeated sequences, was used in combination with a fluorescent complementary DNA to create dense clusters of fluorescence. These clusters, each corresponding to a single target molecule, can be detected unambiguously due to their high signal/noise ratios. The limit of detection of this assay is approximately 1 amol. This simple single molecule assay allows high detection sensitivity without the use of complex equipment.

Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification

We report a sensitive and efficient magnetic bead-based assay for viral DNA identification using isothermal amplification of a reporting probe.

Mycobacterium tuberculosis detection via rolling circle amplification

Hybridization-based assays for DNA detection often use single-stranded DNA (ssDNA) probes to capture ssDNA targets in solution. Unfortunately, these assays are often not able to detect double-stranded DNA (dsDNA). Here, we achieve highly sensitive dsDNA target detection by including short oligonucleotide sequences during denaturing and cooling. After performing an isothermal nucleic acid amplification technique (Rolling Circle Amplification, RCA), these captured dsDNA targets are labeled, allowing single amplified molecules to be imaged and counted. This detection method was first applied to the detection of PCR-generated (polymerase chain reaction) dsDNA targets, yielding a limit of detection of 4.25 fM. As an application of the developed assay, the detection of extracted Mycobacterium tuberculosis (M. tb.) genomic DNA was attempted. A M. tb.-specific target was detected with high specificity compared to similar bacteria, and a detection limit of 10 000 colony forming units (cfu) ml-1 was achieved, close to the sensitivity required for clinical diagnosis.

Harnessing nanotechnology to create new diagnostics and treatments for infectious disease

Infectious microorganisms represent the causes of many of the most common and dangerous diseases in our modern society. Two major problems exist when tackling the problem of infections: detection and treatment. Here we utilize two distinct nanotechnology platforms to combat these problems. First, we demonstrate a novel detection method for identifying Mycobacterium tuberculosis DNA using rolling circle amplification (RCA) of the pathogenic DNA. Second, we design a system for the development of antiviral drug combinations using feedback system control to rapidly determine effective antiviral drug combinations. Using Herpes Simplex Virus (HSV-1) as a model we show the effectiveness of this approach. Taken together, we have begun to develop a platform for better handling of infectious disease in the future using novel nanotechnology and nanomedicine approaches.