Yongxia Zhang

An evaluation of chemical photoreactivity and the relationship to phototoxicity

The existing regulatory guidance for photosafety testing of new drug products states that studies are warranted for those chemicals that both absorb light in the range of 290-700 nm, and that are either applied locally/topically, or reach (EMEA)/significantly partition (FDA) to the skin or eyes. The initial in vitro study recommended for the assessment of phototoxic potential is the 3T3 Neutral Red Uptake (NRU) Assay. The current study was undertaken to establish superior triggers for the initiation of biological photosafety testing. In this study, photophysical and photochemical parameters for 40 drug or drug-like molecules were studied. Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA), and a fivefold cross-validation PLS algorithm were used to evaluate the relationship between subsets of photophysical and photochemical parameters with the 3T3 NRU PIF/MPE (Photo Irritation Factor/Mean Photo Effect) results. The parameters most indicative of a 3T3 NRU positive PIF or MPE score were the extent of degradation in solution, the quantum yield of formation of singlet oxygen and the relative formation of superoxide anion. The results demonstrate that while absorption of light is critical to the induction of a light-induced process, it is the resultant events that may be used to predict the 3T3 NRU assay result. It is therefore proposed that the trigger for photosafety testing be revised to include a molecular basis for photoreactivity. From this limited investigation, estimated thresholds leading to 3T3 NRU positive results due to photodegradation, formation of singlet oxygen quantum yield or a relative superoxide anion formation value are proposed.

Ultra-Fast and Sensitive Detection of Non-Typhoidal Salmonella Using Microwave-Accelerated Metal-Enhanced Fluorescence (“MAMEF”)

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1∶1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).

Development of a Microwave—Accelerated Metal-Enhanced Fluorescence 40 Second, <100 cfu/mL Point of Care Assay for the Detection of Chlamydia Trachomatis

An inexpensive technology to both lyse Chlamydia trachomatis (CT) and detect DNA released from CT within 40 s is demonstrated. In a microwave cavity, energy is highly focused using 100-nm gold films with “bow-tie” structures to lyse CT within 10 s. The ultrafast detection of the released DNA from less than 100 cfu/mL CT is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. This new “ release and detect” platform technology is a highly attractive alternative method for the lysing of bacteria, DNA extraction, and the fast quantification of bacteria and potentially other pathogenic species and cells as well. Our approach is a significant step forward for the development of a point of care test for CT.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: A new Rapid Catch and Signal (RCS) technology

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new Catch and Signal technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color Catch and Signal DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis.

Metal-enhanced chemiluminescence from chromium, copper, nickel, and zinc nanodeposits: Evidence for a second enhancement mechanism in metal-enhanced fluorescence

Over the past decade metal-fluorophore interactions, metal-enhanced fluorescence, have attracted significant research attention, with the technology now becoming common place in life science applications. In this paper, we address the underlying mechanisms of metal-enhanced fluorescence (MEF) and experimentally show using chemiluminescence solutions that MEF is indeed underpinned by two complimentary mechanisms, consistent with the recent reports by Geddes and co-workers [Zhang et al., J. Phys. Chem. C 113, 12095 (2009)] and their enhanced fluorescence hypothesis.

Highly sensitive quantitation of human serum albumin in clinical samples for hypoproteinemia using metal-enhanced fluorescence.

In this paper, we have explored metal-enhanced fluorescence (MEF) of the Human serum albumin indicators: Albumin Blue 580, Merocyanine 540 and Bromophenol Blue in close proximity to silver nano-particles, SiFs, from both buffered and clinical samples. The photostability of the Albumin Blue 580 is shown to be much more prolonged from the SiFs as compared to glass (a control sample), potentially allowing for longer detection times to further improve assay statistics. Our findings suggest the widespread use of nanoparticulate SiFs surfaces for the enhanced detection of HSA, particularly for Hypoproteinemia, where an enhanced assay performance at low protein abundance is required.

Metal-enhanced fluorescence from thermally stable rhodium nanodeposits

Different density rhodium nanoparticulate substrates were fabricated by electron-beam physical vapor deposition in order to study the fluorescence of close-proximity fluorophores to the high thermally stable rhodium nanoparticles. We observed an apparently constant metal-enhanced fluorescence (MEF), when fluorophores were placed in close proximity to rhodium nanoparticles before and after autoclaving of the substrates. Fluorophores with different emission wavelength maxima and free-space quantum yields have also been studied and can undergo different enhancements, a 2.5-fold increase in far-field luminescence was observed from 15 nm Rh films for Tinopal, and up to a 10-fold enhancement was observed for fluorescein. Similarly, the near-field fluorescence enhancement values were estimated to be ∼125 and 500 fold, respectively. Further, the electromagnetic field distributions around different size Rh nanoparticles were simulated using FDTD to understand the wavelength dependence of the e-field. Our findings show that the decay time of fluorophores was not reduced near to the rhodium substrates, suggesting only an enhanced electric field component is the mechanism for fluorescence enhancement.

Metal-enhanced fluorescence exciplex emission.

In this letter, we report the first observation of metal-enhanced exciplex fluorescence, observed from anthracene in the presence of diethylaniline. Anthracene in the presence of diethylaniline in close proximity to Silver Island Films (SIFs) shows enhanced monomer and exciplex emission as compared to a non-silvered control sample containing no silver nanoparticles. Our findings suggest two complementary methods for the enhancement: (i) surface plasmons can radiate coupled monomer and exciplex fluorescence efficiently, and (ii) enhanced absorption (enhanced electric near-field) further facilitates enhanced emission. Our exciplex studies help us to further understand the complex photophysics of the metal-enhanced fluorescence technology.

Mixed-metal substrates for applications in metal-enhanced fluorescence

Over the last decade Metal-Enhanced Fluorescence (MEF) has emerged as the next generation of fluorescence spectroscopy, i.e. near-field fluorescence. However, in contrast to our collective knowledge and understanding of classical far-field fluorescence, we know relatively little. MEF is a consequence of the near-field interactions of fluorophores (dipoles) with the surface plasmons generated in plasmon supporting materials, where the optical properties of the metal afford for a wavelength dependence of MEF. In this paper we show that we are not limited to the properties of the individual metals for MEF, but in fact, surface deposits of mixed metals can create new dephased plasmon resonance bands, not present in the individual metals themselves. Subsequently, mixed metal substrates (MMS) offer significant opportunities for the multifarious and forever growing applications of MEF.

P4-S1.09 Development of a microwave: accelerated metal-enhanced fluorescence 40 s, <100 cfu/ml point of care assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoea

Chlamydia trachomatis (CT) is the most prevalent bacterial sexually transmitted infection (STIs) reported to the Centers for Disease Control and Prevention (CDC). There were 1.2 million cases of chlamydia reported to the CDC in 2008. Neisseria gonorrhoeae (GC) is also one of the most prevalent sexually transmitted infections in men and women. In 2009, there were 301u2008174, cases reported to the CDC, a rate of 99.1 per 100u2008000 populations. The CDC estimates that STIs cost the healthcare system