THz Detection of Biomolecules in Aqueous Environments—Status and Perspectives for Analysis Under Physiological Conditions and Clinical use

Bioanalytical THz sensing techniques have proven to be an interesting and viable tool for the label-free detection and analysis of biomolecules. However, a major challenge for THz bioanalytics is to perform investigations in the native aqueous environments of the analytes. This review recapitulates the status and future requirements for establishing THz biosensing as a complementary toolbox in the repertoire of standard bioanalytic methods. The potential use in medical research and clinical diagnosis is discussed. Under these considerations, this article presents a comprehensive categorization of biochemically relevant analytes that have been investigated by THz sensing techniques in aqueous media. The detectable concentration levels of ions, carbohydrates, (poly-)nucleotides, active agents, proteins and different biomacromolecules from THz experiments are compared to characteristic physiological concentrations and lower detection limits of state-of-the-art bioanalytical methods. Finally, recent experimental developments and achievements are discussed, which potentially pave the way for THz analysis of biomolecules under clinically relevant conditions.

     

Loss of expression or mutations in the p73 tumour suppressor gene are not involved in the pathogenesis of malignant melanomas

BACKGROUND: The authors present their study on oncologic and functional results of supracricoid partial laryngectomies (SPL) performed on 149 patients between January 1984 and December 1995. METHODS: Cricohyoidopexy (CHP) was carried out on 98 patients and cricohyoidoepiglottopexy (CHEP) on 51 patients. The patients were divided into two groups. The first group included those operated on between January 1984 and December 1992 and who therefore had a minimum follow-up period of 3 years. The second group included those operated on after December 1992 and who therefore had a follow-up period of less than 3 years. The statistical evaluation of this second group was carried out using an actuarial method according to Kaplan-Meier. RESULTS: In the first group, survival rate (regarding disease-related deaths) was 94% (95/101), whereas in the second group, survival rate was 95%. There were 9 recurrences in the 149 patients (6.71%), B of which occurred after CHP (6 for tumor [T] and 2 for node [N]) and 1 (for T) after CHEP. Three of the 6 recurrences for T after CHP occurred in the hypopharynx, 2 in the peristomal area, and 1 in the arytenoid area. The only recurrence for T after CHEP occurred in the paraglottic area. Decannulation was carried out in 85.7% of CHP patients (84/98) and in 98% of CHEP patients (50/51). The nasogastric tube was kept in position for an average of 28 days (range, 15-90 days) in the CHP patients and 15 days (range, 9-90 days) in the CHEP patients. Swallowing was excellent; only a small number of patients (n = 21) were forced to assume a particular posture during meals. Phoniatric controls performed on 104 patients also showed adequate speech recovery. CONCLUSIONS: If the indications are applied scrupulously, CHEP is a valid alternative to partial laryngeal surgery and CHP is a possible alternative to total laryngectomy in the treatment of glottic and supraglottic tumors.     

Label-free THz sensing of genetic sequences: towards 'THz biochips'

THz-wave-based approaches for the label-free characterization of genetic material are described. Time-resolved THz spectroscopic analysis of genetic sequences (polynucleotides) demonstrate a distinct complex refractive index in the THz frequency range as a function of the binding state of the analysed DNA sequences. By monitoring THz signals, one can thus infer the binding state of oligo- and polynucleotides, enabling the label-free determination of the genetic composition of target polynucleotides by sensing their binding to known probe molecules. Here we review integrated THz sensing array developments exhibiting high sensitivity and single-base mutation detection capabilities. Recent achievements using functionalized biosensing arrays of high-Q resonators are illustrated.     

Acidified Nitrite Contributes to the Antitumor Effect of Cold Atmospheric Plasma on Melanoma Cells

Cold atmospheric plasma (CAP) is partially ionized gas near room temperature with previously reported antitumor effects. Despite extensive research and growing interest in this technology, active components and molecular mechanisms of CAP are not fully understood to date. We used Raman spectroscopy and colorimetric assays to determine elevated nitrite and nitrate levels after treatment with a MiniFlatPlaster CAP device. Previously, we demonstrated CAP-induced acidification. Cellular effects of nitrite and strong extracellular acidification were assessed using live-cell imaging of intracellular Ca²⁺ levels, cell viability analysis as well as quantification of p21 and DNA damage. We further characterized these observations by analyzing established molecular effects of CAP treatment. A synergistic effect of nitrite and acidification was found, leading to strong cytotoxicity in melanoma cells. Interestingly, protein nitration and membrane damage were absent after treatment with acidified nitrite, thereby challenging their contribution to CAP-induced cytotoxicity. Further, phosphorylation of ERK1/2 was increased after treatment with both acidified nitrite and indirect CAP. This study characterizes the impact of acidified nitrite on melanoma cells and supports the importance of RNS during CAP treatment. Further, it defines and evaluates important molecular mechanisms that are involved in the cancer cell response to CAP.     

MIA ("melanoma inhibitory activity"). Biological functions and clinical relevance in malignant melanoma]

The protein MIA was identified and isolated from the tissue culture supernatant of melanoma cells in vitro by its ability to inhibit thymidine incorporation by melanoma cell lines. After purification and partial sequencing of the peptide, a fragment of the MIA cDNA was cloned by RT-PCR. This cDNA fragment was used to screen phage libraries and subsequently fully encoding human and murine MIA cDNA and genomic DNA clones were obtained. The MIA gene spans a region of approximately 2 kb and is divided into 4 exons. Mapping the MIA gene revealed that the human gene is located on chromosome 19 and the murine gene on chromosome 7. The MIA open reading frame spans 131 (human) or 130 (murine) amino acids. The first 24 (human) or 23 (murine) amino acids represent a signal sequence directing the secretion of MIA into the extracellular compartment. The mature, secreted MIA consists of 107 amino acids and its MW is approximately 11 kDa. Preliminary structural data suggests that MIA is a small globular protein stabilized by two intramolecular disulfide bonds. Expression studies of protein und mRNA levels indicate that MIA is expressed specifically by malignant melanoma cells and chondrocytes. This points to a highly restricted expression pattern which is controlled by the MIA promoter. In addition, MIA provides a clinically useful parameter in patients with malignant melanoma. Enhanced values were measured in the serum of all patients with metastatic melanoma (stage III and IV). In vitro and in vivo experiments using recombinant MIA protein revealed that MIA specifically inhibits attachment of melanoma cells to fibronectin and laminin. Further analysis indicated a direct binding between MIA and the matrix proteins. This finding provides an explanation for the capability of MIA to inhibit proliferation of melanoma cells in vitro. Our studies suggest a putative function of MIA in regulated detachment of melanoma cells from the extracellular matrix which is an important step in metastasis.     

Cold Atmospheric Plasma Changes the Amino Acid Composition of Solutions and Influences the Anti-Tumor Effect on Melanoma Cells

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.     

Integrated planar terahertz resonators for femtomolar sensitivity label-free detection of DNA hybridization

A promising label-free approach for the analysis of genetic material by means of detecting the hybridization of polynucleotides with electromagnetic waves at terahertz (THz) frequencies is presented. Using an integrated waveguide approach, incorporating resonant THz structures as sample carriers and transducers for the analysis of the DNA molecules, we achieve a sensitivity down to femtomolar levels. The approach is demonstrated with time-domain ultrafast techniques based on femtosecond laser pulses for generating and electro-optically detecting broadband THz signals, although the principle can certainly be transferred to other THz technologies.