Special Issue "Bioanalysis in Vivo/in Vitro"

Abstract: Site-specific chemical labeling utilizing small fluorescent molecules is apowerful and attractive technique for in vivo and in vitro analysis of cellular proteins,which can circumvent some problems in genetic encoding labeling by large fluorescentproteins. In particular, affinity labeling based on metal-chelation, advantageous due to thehigh selectivity/simplicity and the small tag-size, is promising, as well as enzymaticcovalent labeling, thereby a variety of novel methods have been studied in recent years.This review describes the advances in chemical labeling of proteins, especially highlightingthe metal-chelation methodology.

Abstract: To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer.Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimerhas a K_d value which is 1/10 of that of the monomers from which it is composed. Also, thedesigned aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers againstvascular endothelial growth factor (VEGF₁₆₅), which is a homodimeric protein. As in thecase of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower K_d value than that of the monomer. This study demonstrated that the dimerization of aptamerseffectively improves the affinities of those aptamers for their targets.

Abstract: Whole-body imaging with fluorescent proteins has been shown to be a powerfultechnology with many applications in small animals. Brighter, red-shifted proteins can makewhole-body imaging even more sensitive due to reduced absorption by tissues and less scatter.For example, a new protein called Katushka has been isolated that is the brightest known proteinwith emission at wavelengths longer than 620 nm. This new protein offers potential for non-invasive whole-body macro imaging such as of tumor growth. For subcellular imaging, toobserve cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in thenucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Thenuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as theytrafficked by various means or adhered to the vessel surface in the abdominal skin flap. Duringextravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cellsexited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasmand nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphaticvessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumormicroenvironment as well as drug response at the cellular level in live animals in real time, wedeveloped a new imageable three-color animal model. The model consists of GFP-expressingmice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highlysensitive small animal imaging system, subcellular dynamics can now be observed in live mice inreal time. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.

Abstract: A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for “focused genomics” that follows “comprehensive genomics”.

Abstract: Study of genetic material allows the comprehension the origin of the many biochemical changes that follow diseases, like cancer, promoting the development of early preventive inquiry and more efficient individual treatments. Raman spectroscopy can be an important tool in DNA study, since it allows probe molecular vibrations of genetic material in a fast way. The present work established a systematic way for extract DNA in suitable concentrations and structural integrity allowing studies by Raman spectroscopy or other spectroscopic technique, including bio-analytical sensors for probing genetic alterations.

Abstract: A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra of blood serum samples from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. Spectra were measured by means of a Raman confocal microspectrometer apparatus using the 632.8 nm line of a He-Ne laser source. A discrete wavelet transform decomposition method has been applied to the recorded Raman spectra in order to overcome problems related to low-level signals and the presence of noise and background components due to light scattering and fluorescence. This numerical data treatment can automatically extract quantitative information from the Raman spectra and makes more reliable the data comparison. Even if an exhaustive investigation has not been done in this work, the feasibility of the follow-up monitoring of pemphigus vulgaris pathology has been clearly proved with useful implications for the clinical applications.

Abstract: Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2^nd and 3^rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold.