Profile for Stephen M. Lane

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the Raman fingerprints of the metabolic states of Escherichia coli ( E. coli ) cells and to determine the spectral changes associated with cellular response to the antibiotic Cefazolin. The Raman spectra of E. coli cells sampled at different time points in the bacterial growth curve exhibited several spectral features that enabled direct identification of the growth phase of the bacteria. Four groups of Raman peaks were identified based on similarities in the time-dependent behavior of their intensities over the course of the growth curve. These groupings were also consistent with the different biochemical species represented by the Raman peaks. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, while protein-specific Raman vibrations increased at different rates. The adenine ring-breathing mode at 729 and the 1245 cm(-1) vibration peaked in intensity within the first 10 h and decreased afterward. Application of principal component analysis (PCA) to the Raman spectra enabled accurate identification of the different metabolic states of the bacterial cells. The Raman spectra of cells exposed to Cefazolin at the end of log phase exhibited a different behavior. The 729 and 1245 cm(-1) Raman peaks showed a slight decrease in intensity from 4 to 10 h after inoculation. Moreover, a shift in the spectral position of the adenine ring-breathing mode from 724 to 729 cm(-1), which was observed during normal bacterial growth, was inhibited during antibiotic drug treatment. These results suggest that potential Raman markers exist that can be used to identify E. coli cell response to antibiotic drug treatment.

Specific, quantitative, and sensitive protein detection with minimal sample preparation is an enduring need in biology and medicine. Protein detection assays ideally provide quick, definitive measurements that use only small amounts of material. Fluorescence cross-correlation spectroscopy (FCCS) has been proposed and developed as a protein detection assay for several years. Here, we combine several recent advances in FCCS apparatus and analysis to demonstrate it as an important method for sensitive, quantitative, information-rich protein detection with low false positives. The addition of alternating laser excitation (ALEX) to FCCS along with a method to exclude signals from occasional aggregates leads to a very low rate of false positives, allowing the detection and quantification of the concentrations of a wide variety of proteins. We detect human chorionic gonadotropin (hCG) using an antibody-based sandwich assay and quantitatively compare our results with calculations based on binding equilibrium equations. Furthermore, using our aggregate exclusion method, we detect smaller oligomers of the prion protein PrP by excluding bright signals from large aggregates.

Imaging the structure and correlating it with the biochemical content of the retina holds promise for fundamental research and for clinical applications. Optical coherence tomography (OCT) is commonly used to image the 3D structure of the retina and while the added functionality of biochemical analysis afforded by Raman scattering could provide critical molecular signatures for clinicians and researchers, there are many technical challenges to combine these imaging modalities. We describe an OCT microscope for ex-vivo imaging combined with Raman spectroscopy capable of collecting morphological and molecular information about a sample simultaneously. We present our first results and discuss the challenges to further development of this dual-mode instrument and limitations for future in-vivo retinal imaging.

We describe an aptamer-based surface enhanced resonance Raman scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human alpha-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

We describe an aptamer-based surface enhanced resonance Raman scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human alpha-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

Currently, a combination of technologies is typically required to assess the malignancy of cancer cells. These methods often lack the specificity and sensitivity necessary for early, accurate diagnosis. Here we demonstrate using clinical samples the application of laser trapping Raman spectroscopy as a novel approach that provides intrinsic biochemical markers for the noninvasive detection of individual cancer cells. The Raman spectra of live, hematopoietic cells provide reliable molecular fingerprints that reflect their biochemical composition and biology. Populations of normal T and B lymphocytes from four healthy individuals and cells from three leukemia patients were analyzed, and multiple intrinsic Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for cancer cell identification. A combination of two multivariate statistical methods, principal component analysis (PCA) and linear discriminant analysis (LDA), was used to confirm the significance of these markers for identifying cancer cells and classifying the data. The results indicate that, on average, 95% of the normal cells and 90% of the patient cells were accurately classified into their respective cell types. We also provide evidence that these markers are unique to cancer cells and not purely a function of differences in their cellular activation.

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific, slow, biologically perturbing, or a combination thereof. Here, we show that single-cell micro-Raman spectroscopy averts these shortcomings and can be used to discriminate between unfixed normal human lymphocytes and transformed Jurkat and Raji lymphocyte cell lines based on their biomolecular Raman signatures. We demonstrate that single-cell Raman spectra provide a highly reproducible biomolecular fingerprint of each cell type. Characteristic peaks, mostly due to different DNA and protein concentrations, allow for discerning normal lymphocytes from transformed lymphocytes with high confidence (p << 0.05). Spectra are also compared and analyzed by principal component analysis to demonstrate that normal and transformed cells form distinct clusters that can be defined using just two principal components. The method is shown to have a sensitivity of 98.3% for cancer detection, with 97.2% of the cells being correctly classified as belonging to the normal or transformed type. These results demonstrate the potential application of confocal micro-Raman spectroscopy as a clinical tool for single cancer cell detection based on intrinsic biomolecular signatures, therefore eliminating the need for exogenous fluorescent labeling.

Surface-enhanced Raman scattering (SERS) intensities for individual Au nanospheres, nanoshells, and nanosphere and nanoshell dimers coated with nonresonant molecules are measured, where the precise nanoscale geometry of each monomer and dimer is identified through in situ atomic force microscopy. The observed intensities correlate with the integrated quartic local electromagnetic field calculated for each specific nanostructure geometry. In this study, we find that suitably fabricated nanoshells can provide SERS enhancements comparable to nanosphere dimers.

We present the development of nanoscale pH sensors based on functionalized silver nanoparticles and surface-enhanced Raman scattering (SERS). The SERS spectrum from individual silver nanoparticle (50-80 nm in diameter) clusters functionalized with 4-mercaptobenzoic acid shows a characteristic response to the pH of the surrounding solution and is sensitive to pH changes in the range of 6-8. Measurements from nanoparticles incorporated in living Chinese hamster ovary cells demonstrate that the nanoparticle sensors retain their robust signal and sensitivity to pH when incorporated into a cell.

Bipyridine ligands containing pendant methyl, amino, and amino-boronic acid groups were synthesized. Coordination complexes of these ligands with rhenium were prepared straightforwardly and in good yield. The fluorescence behavior of the Re complexes was studied as a function of pH and exposure to various concentrations of glucose. The methyl bipyridine complex showed no change in fluorescence with pH, the amino derivative showed a rapid decrease from low pH to neutral, and the amino-boronate derivative showed little change from pH 4 to 10. Fluorescence quenching was observed at high pH as expected on the basis of a photoinduced electron transfer (PET) signaling mechanism. This behavior can be explained on the basis of the first oxidation and reduction potentials of these complexes. Glucose testing showed a significant dependence on the solvent system used. In pure methanol, the rhenium boronate complex exhibited a 55% fluorescence intensity increase upon increasing glucose concentration from 0 to 400 mg/dL. However, in 50 vol % methanol/phosphate buffered saline, none of the complexes showed significant response in the glucose range of physiological interest.