Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

New indodicarbocyanine dyes for the biological microchip technology

New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.     

New indodicarbocyanine dyes for the biological microchip technology

New indodicarbocyanine dyes with the carboxybutyl group in position 3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.     

Optical properties of fluorochromes promising for use in biological microchips

Fourteen fluorochromes (free and oligonucleotide-bound) and five ligands commonly used to quantitatively assess DNA duplexes or complexes with proteins in microchips were studied by measuring their fluorescence excitation and emission spectra. The spectral changes are described that were caused by oligonucleotide binding, solution hybridization, or varying the temperature. The data are discussed from the standpoint of applicability and limitations of the fluorochromes and the ligands in qualitative and quantitative assays for DNA duplexes in microchips.     

Analysis of NAT2 point mutations with biological microchips

The NAT2 product, N-acetyltransferase 2, is involved in biotransformation and detoxification of several aromatic amines (in particular, 2-aminofluorene, 4-aminobiphenyl, and 4-naphthylamine), which are strongly mutagenic and carcinogenic, and acetylates some drugs, affecting their metabolism. A biological microchip was developed to detect 16 point mutations, which determine 36 alleles and 660 genotypes of NAT2. The genotypes can be divided into four groups according to the acetylator phenotype: groups with rapid (R/R), intermediate (R/S), or slow (S/S) acetylation and a group combining intermediate and slow alleles (“R/S or S/S”). The last group includes the alleles determined by combinations of seven mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, and 857G/A), whose cis or trans position is detectable by restriction enzyme analysis. The NAT2 genotype was unequivocally established for 37 out of 71 DNA specimens, while the other 34 specimens were characterized by more than two genotypes. By the acetylator phenotype, 16 out of the 34 genotypes were assigned to the group “R/S or S/S,” combining mutations 282C/T, 341T/C, 481C/T, 590G/A, and 803A/G. Thus, the biochip allows primary analysis of most NAT2 polymorphic substitutions, the acetylator genotype being important to know in predictive medicine and individualized therapy.     

Identification of functionally significant mutations in NAT2 gene using biological microchips

The product of gene NAT2 (N-acetyltransferase 2) is involved in the biotransformation system and participates in detoxication of some arylamine derivatives (in particular 2-aminofluorene, 4-aminobiphenyl and 4-naphthylamine) which are strongly mutagenic and carcinogenic. It also renders toxicological and pharmacological influence on a metabolism of medical products metabolized by the enzyme. We developed a microchip for detection of 16 functionally significant mutations coding 36 alleles of gene NAT2. Combinations of these alleles allow us to reveal more than 660 genotypes, which can be divided into four groups according acetylation phenotype: "fast" (R/R), "intermediate" (R/S), "slow" (S/S) and group with average or slow acetylating (R/S or S/S) alleles. The groups "R/S or S/S" include alleles, formed by a combination of 7 mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, 857G/A), theirs cis-trans position can be revealed by restriction analysis. In 37 of 71 DNA samples we unequivocally defined NAT2-genotypes, and other 34 samples have been characterized by more than two genotypes. 16 samples out of 34 had acetylation phenotype of group "R/S or S/S", which is characterized by the following combination of mutations: 282C/T, 341T/C, 481C/T, 590G/A and 803A/G. Thus, the developed biochip is a convenient screening method for primary detection of the majority of polymorphic replacements in gene NAT2.     

Synthesis and biological properties of galactofuranosyl-containing fluorescent dyes

Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.     

Silicon microchips for manipulating cell-cell interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation. In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions. In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling (1). Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-microm gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.     

Nanostructured optical microchips for cancer biomarker detection

Herein we report the label-free detection of a cancer biomarker using newly developed arrayed nanostructured Fabry-Perot interferometer (FPI) microchips. Specifically, the prostate cancer biomarker free prostate-specific antigen (f-PSA) has been detected with a mouse anti-human PSA monoclonal antibody (mAb) as the receptor. Experiments found that the limit-of-detection of current nanostructured FPI microchip for f-PSA is about 10pg/mL and the upper detection range for f-PSA can be dynamically changed by varying the amount of the PSA mAb immobilized on the sensing surface. The control experiments have also demonstrated that the immunoassay protocol used in the experiments shows excellent specificity and selectivity, suggesting the great potential to detect the cancer biomarkers at trace levels in complex biofluids. In addition, given its nature of low cost, simple-to-operation and batch fabrication capability, the arrayed nanostructured FPI microchip-based platform could provide an ideal technical tool for point-of-care diagnostics application and anticancer drug screen and discovery.     

Near infrared dyes as lifetime solvatochromic probes for micropolarity measurements of biological systems

The polarity of biological mediums controls a host of physiological processes such as digestion, signaling, transportation, metabolism, and excretion. With the recent widespread use of near-infrared (NIR) fluorescent dyes for biological imaging of cells and living organisms, reporting medium polarity with these dyes would provide invaluable functional information in addition to conventional optical imaging parameters. Here, we report a new approach to determine polarities of macro- and microsystems for in vitro and potential in vivo applications using NIR polymethine molecular probes. Unlike the poor solvatochromic response of NIR dyes in solvents with diverse polarity, their fluorescence lifetimes are highly sensitive, increasing by a factor of up to 8 on moving from polar to nonpolar mediums. We also established a correlation between fluorescence lifetime and solvent orientation polarizability and developed a lifetime polarity index for determining the polarity of complex systems, including micelles and albumin binding sites. Because of the importance of medium polarity in molecular, cellular, and biochemical processes and the significance of reduced autofluorescence and deep tissue penetration of light in the NIR region, the findings reported herein represent an important advance toward using NIR molecular probes to measure the polarity of complex biological systems in vitro and in vivo.     

Standardization of biological dyes and stains: pitfalls and possibilities

Summary The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.This paper is dedicated to my academic teacher, Prof. Dr. D.H. Wittekind, on the occasion of his 70th birthday     

Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging

A simple one-pot-procedure for preparation of protein-reactive, water-soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro- and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato group directly on the dye fluorophore system, dyes with high fluorescence quantum yields in water were generated. Both iodo- and chloroacetamido derivatives were shown to be useful in protein labeling. Less reactive chloroacetamides will be preferential for selective labeling of the most reactive cysteines.     

Protein microchips: use for immunoassay and enzymatic reactions

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-microm gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.     

Standardization of biological dyes and stains: pitfalls and possibilities.

The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.     

Effect of mixing on reaction-diffusion kinetics for protein hydrogel-based microchips

Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction-diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction-diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics.     

Effects of various fluorochromes and competition between labeled oligonucleotides on their hybridization to oligonucleotides immobilized on biological microchips

Some technical tips are described on how to improve the discrimination between perfect and imperfect duplexes formed by hybridization of fluorescently labeled oligonucleotides to biological microchips. Model experiments were performed to assess the precision of the method. Effects of labeling on the efficiency of hybridization and some properties of competitive hybridization were studied using short synthetic oligonucleotides and three most popular fluorochromes as examples.     

Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.