Colloidal semiconductor quantum dots (QDs) seem suitable for labeling certain biomolecules for use in fluorescent tagging applications, such as fluoro-immunoassays. Compared to organic dye labels, Qds are resistant to photo-degradation, and these luminescent nanoparticles have size-dependent emission spectra spanning a wide range of wavelengths in the visible and near IR. We previously described an electrostatic self-assembly approach for conjugating highly luminescent colloidal CdSe-ZnS core-shell Qds with engineered two-domain recombinant proteins. Here we describe the application of this approach to prepare QD conjugates with the (Beta) 2 immunoglobin G (IgG) binding domain of streptococcal protein G (PG) appended with a basic lucine zipper attachment domain (PG-zb). We also demonstrate that the QD/PG conjugates retain their ability to bind IgG antibodies, and that a specific antibody coupled to QD via the PG functional domain efficiently binds its antigen. These preliminary results indicate that electrostatically self-assembled QD/PG-zb/IgG bioconjugates can be used in fluoro-immunoassays.

A new method for in vitro and possibly in vivo ultrahigh-resolution colocalization and distance measurement between biomolecules is described, based on semiconductor nanocrystal probes. This ruler bridges the gap between FRET and far-field (or near-field scanning optical microscope) imaging and has a dynamic range from few nanometers to tens of micrometers. The ruler is based on a stage-scanning confocal microscope that allows the simultaneous excitation and localization of the excitation point-spread-function (PSF) of various colors nanocrystals while maintaining perfect registry between the channels. Fit of the observed diffraction and photophysics-limited images of the PSFs with a two-dimensional Gaussian allows one to determine their position with nanometer accuracy. This new high-resolution tool opens new windows in various molecular, cell biology and biotechnology applications.

The development of new fluorescent probes has impacted many areas of research such as medical diagnostics, high-speed drug screening, and basic molecular biology. Main limitations to traditional organic fluorophores are their relatively weak intensities, short life times (eg., photobleaching), and broad emission spectra. The desire for more intense fluorescent probes with higher quality photostability and narrow emission wavelengths has led to the development and utilization of semiconductor quantum dots as a new label. In this work, we have modified semicondutor quantum dots (QD's) with synthetic oligonucleotides to probe a specific DNA target sequence both in solution as well as immobilized on a solid substrate. In the first approach, specific target sequences are detected in solution by using short oligonucleotide probes, which are covalently linked to semiconductor quantum dots. In the second approach, DNA target sequences are covalently attached to a glass substrate and detected using oligonucleotides linked to semiconductor quantum dots.

The 1 - 100 nanometer size range encompasses the dimensions of proteins and DNA. In this size range the bulk properties of inorganic materials become influenced by quantum mechanical effects and become size-dependent. Semiconductor nanoparticles are photoluminescent throughout the visible; the emission maximum is dictated by particle size, nature of the surface, and nature of the bulk material. We have used the photoluminescence of semiconductor nanoparticles to infer how oligonucleotides with unusual structure bind to the nanoparticles, providing insight into local structure and flexibility of the DNA. More recently we have examined the effects of base modifications on these binding events. Metallic nanoparticles can also interact with DNA, and these interactions can be monitored by the visible absorbance spectrum of the nanoparticles and by surface-enhanced Raman spectroscopy (SERS). Metallic surfaces that are rough on the nanometer scale are known to enhance the Raman signals of adsorbates by up to a million-fold. The result of photoluminescence titrations of abasic DNA and SERS DNA-nanoparticle studies will be reported.

We report experimental and theoretical results on the effect of electromagnetic coupling between metal particles in surface-enhanced Raman scattering (SERS). Model calculations of the near-field optical properties of Ag and Au nanoparticle-aggregates show that the electromagnetic surface-enhancement factor can reach 11 orders-of-magnitude in gaps between nearly touching particles. Single particles exhibit a much weaker enhancement, unless the particles contain extremely sharp surface protrusions. Data on spectral fluctuations in single-molecule SERS and measurements on the efficiency of nanofabricated SERS substrates give experimental support for the idea that an efficient interparticle coupling is a necessary requirement for an ultra-high surface-enhancement. We suggest a route for biorecognition induced coupling of metal particles for use in biosensing applications.

Recent results suggest that surface-enhanced Raman Spectroscopy (SERS) of single adsorbate molecules is possible under appropriate circumstances. We propose that this phenomenon is associated with very intense enhancements available at interstitial sites (hot spots) of nanoparticle assemblies (either colloid particle aggregates or rough surfaces) illuminated with light of an appropriate wavelength so as to excite surface plasmons, coupled with additional resonance enhancements due to a judicious choice of ad-molecule. The former contribution, known as electromagnetic (EM) enhancement, has been known for years to be capable of producing EM hot spots where the enhancement can top 10¹¹. This fact seems to have been rediscovered recently. It is also known that the fields at the surface of fractal aggregates commonly show hot spots. These are also, at times, capable of such high local enhancements. On fractals, the location of these hot spots are, however, highly dependent on parameters such as the excitation wavelength. In contrast, small compact clusters (when properly designed) have the benefit of a wavelength-independent hot spot where a small number of molecules could be (chemically) directed and detected. This insight suggests an eventual optimally engineered single-molecule SERS system with predictable enhancement capabilities and optimal adsorption (i.e. chemical) characteristics at the hot spot.

In the vicinity of small metal particles in dimensions of tens of nanometers or close to nanostructured metallic surfaces, local optical fields can be strongly enhanced due to resonances with collective excitation of the conduction electrons in these metallic nanostructures. These enhanced local fields open up exciting opportunities for enhancing spectroscopic signals and to perform spectroscopy on single molecules. We report surface-enhanced Raman studies on silver and gold nanostructures. The field strengths in the hot spots on silver and gold colloidal cluster structures are inferred to be enhanced on the order of 10³ resulting in field enhancement factors for Raman scattering up to 10¹². Simultaneously, as a further advantage for Raman spectroscopy, the fluorescence of the target molecules is quenched by new non-radiative decay channels to the metal.

Using wide-field illumination, optically active 'hot' particles can be screened from a heterogeneous colloid using a near-IR excitation source. Atomic force microscopy (AFM) correlated with surface-enhanced Raman scattering (SERS) measurements reveal that the majority of these particles are small nanoparticle aggregates. This finding indicates a strong dependence between particle size and Raman enhancement. Furthermore, these 'hot' nanoaggregates display an intermittent on-off emission behavior similar to 'blinking' SERS exhibited at 488 nm and 514 nm laser excitation. This behavior, not observed in bulk SERS studies, can only be examined at the single particle level because of variations in particle size, shape, and surface defects. Further examination at the single particle level using a near-IR excitation source could led to new insights regarding the fundamental nature of 'hot' particles as well as the SERS mechanism.

We report on the study of the photodecomposition of single Rhodamine 6G (R6G) dye molecules adsorbed on silver nanoparticles. The nanoparticles were immobilized and spatially isolated on polylysine-derivatized glass coverslips, and confocal laser microspectroscopy was used to obtain surface-enhanced Raman scatters (SERS) spectra from individual R6G molecules. The photodecomposition of these molecules was observed with 150-ms temporal resolution. The photoproduct was identified as graphitic carbon based on the appearance of bread SERS vibrational bands at 1592 cm^-1 and 1340 cm^-1 observed in both bulk and averaged single-molecule photoproduct spectra. In contrast, when observed at the single-molecule level, the photoproduct yielded sharp SERS spectra. The inhomogeneous broadening of the bulk SERS spectra is due to a variety of photoproducts in different surface orientations and is a characteristic of ensemble-averaged measurement of disordered systems. These single-molecule studies indicate a photodecomposition pathway by which the R6G molecule desorbs from the metal surface, an excited-state photoreaction occurs, and the R6G photoproduct(s) readsorbs to the surface. A SERS spectrum is obtained when either the intact R6G or the R6G photoproduct(s) are adsorbed on a SERS-active site. This work further illustrates the power of single-molecule spectroscopy (SMS) to reveal unique behaviors of single molecules that are not discernable with bulk measurements.

Surface-enhanced resonance Raman scattering (SERRS) is a very sensitive and selective detection method that can be used for the analysis of both DNA and P-450s. A number of factors have limited the broader application of the technique. These limitations are described and addressed. An approach to reduce the problems associated with variation of the silver colloids used to provide surface enhancement and chemical methodologies that ensure surface adsorption are presented. A practical approach was used to investigate the nature of the effect. This approach has highlighted the importance of resonance enhancement for ultimate sensitivity. Two approaches to achieve successful detection of DNA using SERRS are described, and, using these two approaches, the possibility of multiplexing is also demonstrated. The analysis of proteins by SERRS is discussed and P-450 is presented as a specific example of the information that may be gained from SERRS of proteins.

In recent years there has been a great deal of interest in the measurement of DNA hybridization at surfaces. Surface-confined DNA hybridization has been used to monitor gene expression, to detect the presence of a particular DNA sequence and determine single nucleotide polymorphisms (SNPs). DNA microarrays, which can contain thousands of discrete DNA sequences on a single surface, have become widely used for hybridization studies. While a powerful technique, this technology is limited by the stability of the fluorescent dyes used to label the DNA, and the need to perform measurements ex-situ to reduce the fluorescence background. In this report, we describe the use of colloid-amplified surface plasmon resonance (SPR) to measure DNA hybridization at surfaces. SPR is a surface sensitive technique, which can be used to study hybridization in situ, and the use of colloidal metal tags provides excellent sensitivity. Angle-scanning SPR has been used to study oligonucleotide hybridization to surface confined probes, and work is underway to apply SPR imaging to study DNA hybridization in macro- and microarray formats.

Sunlight can have deleterious effects on humans: causes sunburns and is the principal cause of skin cancers. Usage of TiO₂ (and ZnO) in sunscreen lotions, widely used as UVA/UVB blockers, and intended to prevent sunburns and to protect consumers from skin cancers (carcinomas and melanomas) is examined. Although used to mineralize many undesired organic pollutants, TiO₂ is considered to be a safe physical sunscreen agent because it reflects and scatters both UVB (290-320 nm) and UVA (320-400 nm) sunlight; however, it also absorbs substantial UV radiation which, in aqueous media, yields hydroxyl radial ((DOT)OH) species. These species cause substantial damage to DNA (J. Photochem.Photobio.A:Chem.,111(1997)205). Most importantly, sunlight-illuminated sunscreen TiO₂ particles catalyze DNA damage both in vitro and in human cells (FEBS Letters, 418 (1997)87). These results raise concerns on the overall effects of sunscreens and raise the question on the suitability of photoactive TiO₂ as a sunscreen component without further studies. The photocatalytically active nature of these metal oxides necessitates some changes since even the TiO₂ specimens currently used in suncreams cause significant DNA strand breaks.

Cascade Blue, Sulforhodamine G and yeast alcohol dehydrogenase were encased inside nano-sized silicate shells and their absorption and fluorescence spectrophotometric properties, and the enzyme activity investigated. The stabilized molecules have potential in biosensors, drug delivery, and as recyclable catalysts. Cascade Blue and Sulforhodamine G were attached to 85 nm diameter colloidal gold, encased with silicate, and the gold core dissolved. Fluorescence quenched by the gold was recovered for both dyes, but the peak emission was red-shifted from that in water for Cascade Blue and blue-shifted for Sulforhodamine G. The excitation spectra of these dyes showed similar shifts, presumably reflecting their interaction with the shell interior. The spectrofluorometric results for alcohol dehydrogenase bound to 15 nm diameter colloidal gold were similar. The substrate ethanol and cofactor NAD were permeable to the silicate shell. Only 20% of enzyme activity of ADH was lost after binding to gold, and additional 20% lost by encasing with silicate. Subsequent rate of loss of activity was significantly lowered. This study demonstrated dyes and enzymes can be encased within silicate shells. Whether the shell protects these molecules from the environment, and how the thickness of silicate shells affects the rate of enzyme reactions remains to be investigated.