Minimally Invasive Skin Transcriptome Extraction Using a Dermal Biomarker Patch.

INTRODUCTION: Advances in the scientific understanding of the skin and characteristic genomic dermal signatures continue to develop rapidly. Nonetheless, skin diagnosis remains predicated on a subjective visual examination, frequently followed by biopsy and histology. These procedures often are not sufficiently sensitive, and in the case of many inflammatory diseases, biopsies are not justified, creating a situation where high-quality samples can be difficult to obtain. The wealth of molecular information available and the pace at which new data are acquired suggest that methods for minimally invasive biomarker collection could dramatically alter our understanding of skin disease and positively impact treatment paradigms. METHODS: A chemical method was optimized to covalently modify custom dermal patches with single-stranded DNA that could bind to messenger RNA. These patches were applied to ex vivo skin samples and penetration evaluated by histological methods. Patches were then applied to both the skin of normal human subjects (lower arm) as well as lesional skin of psoriasis patients, and the transcriptome captured (N = 7; 33 unique samples). Standard RNA-Seq processing was performed to assess the gene detection rate and assessments made of the reproducibility of the extraction procedure as well as the overlap with matched punch biopsy samples from the same patient. RESULTS: We have developed a dermal biomarker patch (DBP) designed to be minimally invasive and extract the dermal transcriptome. Using this platform, we have demonstrated successful molecular analysis from healthy human skin and psoriatic lesions, replicating the molecular information captured with punch biopsy. CONCLUSION: This DBP enables an unprecedented ability to monitor the molecular "fingerprint" of the skin over time or with various interventions, and generate previously inaccessible rich datasets. Furthermore, use of the DBP could be favored by patients relative to biopsy by limiting pain resulting from biopsy procedures. Given the large dynamic range observed in psoriatic skin, analysis of complex phenotypes is now possible, and the power of machine-learning methods can be brought to bear on dermatologic disease.

An intercalator film as a DNA-electrode interface.

DNA-surface conjugation is achieved through an intercalating molecular wire, resulting in more efficient electron transfer relative to systems utilizing conventional insulating tethers.

Nucleotide-directed growth of semiconductor nanocrystals.

We engineer colloidal quantum dot nanocrystals through the choice of biomolecular ligands responsible for nanoparticle nucleation, growth, stabilization, and passivation. We systematically vary the presence of, and thereby elucidate the role of, phosphate groups and a multiplicity of functionalities on the mononucleotides used as ligands. The results provide the basis for synthesis of nanoparticles using precisely controlled synthetic oligonucleotide sequences.

DNA-directed synthesis of zinc oxide nanowires on carbon nanotube tips.

This paper describes a class of three component hybrid nanowires templated by DNA directed self-assembly. Through the modification of carbon nanotube (CNT) termini with synthetic DNA oligonucleotides, gold nanoparticles are delivered, via DNA hybridization, to CNT tips that then serve as growth sites for zinc oxide (ZnO) nanowires. The structures we have generated using DNA templating represent an advance toward building higher order sequenced one dimensional nanostructures with rational control.

Amplified electrocatalysis at DNA-modified nanowires.

Arrayed gold nanowires are a novel and useful platform for electrochemical DNA detection. Pilot studies testing the use of these templated structures with an electrocatalytic reporter system revealed that very low detection thresholds for target DNA sequences can be obtained. One factor contributing to the heightened sensitivity is the high signal-to-noise ratio achieved with the large electrocatalytic signals observed at DNA-modified nanowires. Here, we explain the improved sensitivity with evidence illustrating that electrocatalysis at DNA-modified nanostructures generates amplified signals that are significantly larger than those observed at bulk gold surfaces. The results presented strongly suggest that the three-dimensional architectures of the nanowires facilitate the electrocatalytic reaction because of enhanced diffusion occurring around these structures. Effects unique to the nanoscale are shown to underlie the utility of nanowires for DNA biosensing.

Site-specific assembly of DNA and appended cargo on arrayed carbon nanotubes.

We describe a strategy that permits discrete regions of arrayed carbon nanotubes (CNTs) to be functionalized simultaneously and specifically with DNA oligonucleotides. The different chemical properties of two regions on single CNTs and orthogonal chemical coupling strategies have been exploited to derivatize CNTs within highly ordered arrays with multiple DNA sequences. Through duplex hybridization, we then targeted different DNA sequences with appended metal nanoparticles to distinct sites on the CNT architecture with precise spatial control. The materials generated from these studies represent the first CNTs with bipartite functionalization. The approach described provides a high level of precision in parallel and directed assembly of DNA sequences and appended cargo and is useful for the preparation of novel hybrid bionanomaterials.

Ultrasensitive electrocatalytic DNA detection at two- and three-dimensional nanoelectrodes.

Electrochemical DNA detection systems are an attractive approach to the development of multiplexed, high-throughput DNA analysis systems for clinical and research applications. We have engineered a new class of nanoelectrode ensembles (NEEs) that constitute a useful platform for biomolecular electrochemical sensing. High-sensitivity DNA detection was achieved at oligonucleotide-functionalized NEEs using a label-free electrocatalytic assay. Attomole levels of DNA were detected using the NEEs, validating the promise of nanoarchitectures for ultrasensitive biosensing.

Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers.

The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens. A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization. Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H. pylori protein and one that represents a small portion of the 23S rRNA from this organism. Both sequences can be monitored into the nanomolar concentration range. Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity. In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences. An A2143C substitution within the H. pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence. The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed. The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.