The application of a liquid/liquid microsegmented flow for serial high-throughput microanalytical systems shows promising prospects for applications in clinical chemistry, pharmaceutical research, process diagnostics, and analytical chemistry. Microscopy and microspectral analytics offer powerful approaches for the analytical readout of droplet based assays. Within the generated segments, individuality and integrity are retained during the complete diagnostic process making the approach favored for analysis of individual microscaled objects like cells and microorganisms embedded in droplets. Here we report on the online application of surface-enhanced micro-Raman spectroscopy for the detection and quantization of analytes in a liquid/liquid segmented microfluidic system. Data acquisition was performed in microsegments down to a volume of 180 nl. With this approach, we overcome the well-known problem of adhesion of colloid/analyte conjugates to the optical windows of detection cuvettes, which causes the so-called "memory effect". The combination of the segmented microfluidic system with the highly sensitive SERS technique reaches in a reproducible quantification of analytes with the SERS technique.

We examined hair from 15 patients with trichothiodystrophy (TTD), a rare inherited disorder with brittle, cystine-deficient hair. They had a wide variety of phenotypes, from brittle hair only to severe intellectual impairment and developmental delay. Polarizing light microscopic examination showed alternating light and dark (tiger tail) bands under polarizing microscopy. Confocal microscopy captured structural features of breaks in intact TTD hairs. The autofluorescent appearance was regular and smooth in normal donors and markedly irregular in sections of TTD hairs possibly reflecting abnormalities in melanin distribution. Scanning electron microscopy revealed numerous surface irregularities. All TTD hair samples had reduced sulfur content. We observed an inverse correlation (R(val)=0.9) between sulfur content and percent of hairs with shaft abnormalities (trichoschisis, trichorrhexis nodosa, or ribbon/twist). There was no association between clinical disease severity and percent of abnormal hairs. Raman spectra of hairs from TTD patients and normal donors revealed a larger contribution of energetically less favored disulfide conformers in TTD hairs. Our data indicate that the brittleness of the TTD hair is dependent upon abnormalities at several levels of organization. These changes make TTD hairs excessively prone to breakage and weathering.

Raman spectra of normal hair shafts and hair shafts from patients exhibiting trichothiodystrophy (TTD) were obtained using line focus laser illumination. Because hair from TTD patients has a significant decrease in the content of the sulfur-containing amino acids in comparison to normal hair, the 550-500 cm(-1) disulfide stretching mode region of the Raman spectrum was examined in detail. A quantitative spectral analysis demonstrates significant increases in the two energetically less favored gauche-gauche-trans (g-g-t) and trans-gauche-trans (t-g-t) forms. These observations suggest that the increased amounts of these less stable disulfide conformers are contributing factors to or associated with the hair brittleness observed for this congenital disorder. Structure-spectra correlations for the three dominant disulfide conformers are confirmed by quantum chemical calculations using modern density functional theory (DFT).