Jennifer S. Martinez

A complementary palette of fluorescent silver nanoclusters.

We report the synthesis and photophysical properties of silver-nanoclusters templated on DNA, with fluorescence excitation and emission at distinct wavelengths that are tuned to common laser excitation wavelengths.

A Fluorescence Light-Up Ag Nanocluster Probe that Discriminates Single-Nucleotide Variants by Emission Color

Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individuals genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a different alignment between the Ag nanocluster and the enhancer. This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Samples with single-nucleotide variations can be easily identified by the naked eye under UV excitation, making this method a reliable and low-cost assay with a simple readout format.

A DNA-templated fluorescent silver nanocluster with enhanced stability

We report the discovery of a DNA sequence that templates a highly stable fluorescent silver nanocluster. In contrast to other DNA templated silver nanoclusters that have a relatively short shelf-life, the fluorescent species templated in this new DNA sequence retains significant fluorescence for at least a year. Moreover, this new silver nanocluster possesses low cellular toxicity and enhanced thermal, oxidative, and chemical stability.

Waveguide-Based Biosensors for Pathogen Detection

Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave—the evanescent field—whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, “dirty” biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning’s EPIC® Ô, SRU Biosystems’ BIND™, Zeptosense®, etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing.

Ag K-edge EXAFS analysis of DNA-templated fluorescent silver nanoclusters: insight into the structural origins of emission tuning by DNA sequence variations.

DNA-templated silver nanoclusters are promising biological fluorescence probes due to their useful fluorescence properties, including tunability of emission wavelength through DNA template sequence variations. Ag K-edge EXAFS analysis of DNA-templated silver nanoclusters has been used to obtain insight into silver nanocluster bonding, size, and structural correlations to fluorescence. The results indicate the presence of small silver nanoclusters (<30 silver atoms) containing Ag-Ag bonds and Ag-N/O ligations to DNA. The DNA sequence used leads to differences in silver-DNA ligation as well as silver nanocluster size. The results support a model in which cooperative effects of both Ag-DNA ligation and variations in cluster size lead to the tuning of the fluorescence emission of DNA-templated silver nanoclusters.

Mammalian Stem Cells Reprogramming in Response to Terahertz Radiation

We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma (PPARG). Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming.

A Hybrid DNA-Templated Gold Nanocluster For Enhanced Enzymatic Reduction of Oxygen

We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ∼1 nm in diameter and possessing ∼7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ∼15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e(-) reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

Specificity and Heterogeneity of Terahertz Radiation Effect on Gene Expression in Mouse Mesenchymal Stem Cells

We report that terahertz (THz) irradiation of mouse mesenchymal stem cells (mMSCs) with a single-frequency (SF) 2.52 THz laser or pulsed broadband (centered at 10 THz) source results in irradiation specific heterogenic changes in gene expression. The THz effect depends on irradiation parameters such as the duration and type of THz source, and on the degree of stem cell differentiation. Our microarray survey and RT-PCR experiments demonstrate that prolonged broadband THz irradiation drives mMSCs toward differentiation, while 2-hour irradiation (regardless of THz sources) affects genes transcriptionally active in pluripotent stem cells. The strictly controlled experimental environment indicates minimal temperature changes and the absence of any discernable response to heat shock and cellular stress genes imply a non-thermal response. Computer simulations of the core promoters of two pluripotency markers reveal association between gene upregulation and propensity for DNA breathing. We propose that THz radiation has potential for non-contact control of cellular gene expression.

Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.

Non-thermal effects of terahertz radiation on gene expression in mouse stem cells

Abstract In recent years, terahertz radiation sources are increasingly being exploited in military and civil applications. However, only a few studies have so far been conducted to examine the biological effects associated with terahertz radiation. In this study, we evaluated the cellular response of mesenchymal mouse stem cells exposed to THz radiation. We apply low-power radiation from both a pulsed broad-band (centered at 10 THz) source and from a CW laser (2.52 THz) source. Modeling, empirical characterization, and monitoring techniques were applied to minimize the impact of radiation-induced increases in temperature. qRT-PCR was used to evaluate changes in the transcriptional activity of selected hyperthermic genes. We found that temperature increases were minimal, and that the differential expression of the investigated heat shock proteins (HSP105, HSP90, and CPR) was unaffected, while the expression of certain other genes (Adiponectin, GLUT4, and PPARG) showed clear effects of the THz irradiation after prolonged, broad-band exposure.