Biophysical methods for biochip analysis. Use of wide-field digital fluorescence microscopy

This paper discusses the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitivity throughput recording of analyses with biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides a higher rate of the analysis compared with laser scanning devices. With this analyzer, the scope where biological microchips can be applied becomes wider, development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for performing routine analyses can be reduced.     

Adsorption of avidin on microfabricated surfaces for protein biochip applications

The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radiofrequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that a simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.     

Fluorescent semiconductor nanocrystals (quantum dots) in protein biochips

Understanding the biological processes in cells, tissues, and organisms requires the identification and analysis of multiple biological objects and the mechanisms of their functioning and regulation. The biological chip (biochip) technique is one of the most efficient tools for these tasks. Biochips are highly efficient and can quantitatively register multiple molecules simultaneously in samples of microscopic volume. Biochips allow the parallel genomic or proteomic analysis of normal or pathologically modified cells and tissues and a comparative analysis to elucidate disease-related changes. Fluorescent dyes used for signal readout from biochips have the following disadvantages: low photostability, low brightness, and the presence of a fluorescent background. It was recently shown that these limitations can be removed if fluorescent semiconductor nanocrystals (quantum dots) are used. Individual quantum dots in the form of colloid nanocrystals (QDs) are easily registered by conventional microscopic equipment due to their high brightness; they are extremely resistant to photobleaching and provide unique opportunities for multiplexing. QDs are ideal fluorophores for information readout from biochips and allow for the detection of single molecules. The present work is aimed at developing approaches for the use of QDs in biochip-based detection systems. The possibilities of using QDs in both planar (or matrix) biochips and suspension (or liquid) biochips, which are undergoing intensive development, are demonstrated. The use of the latter in analytical systems for the simultaneous identification of multiple objects in proteomics, genomics, drug testing, and clinical diagnostics is currently increasing. These systems are based on spectrally coded elements (usually polymer microspheres). An advantage of liquid biochips over matrix planar solid biochips is the possibility of the free movement of microspheres in three-dimensional space. Organic fluorophores allow the realization of a limited number of codes, i.e., objects analyzed simultaneously (multiplexing), while semiconductor QDs make possible a significant increase in both biochip multiplexing and the photostability and sensitivity of the biochips. In addition, the use of FRET (Foerster resonance energy transfer) in liquid biochips makes possible an increase in the detection specificity. The absence of a background signal from the fluorescent labels not bound to the microparticles increases the sensitivity of the analysis and provides additional opportunities for multiplex analysis and diagnostics. Thus, a combination of the biochip technique and semiconductor QDs makes it possible to increase the method’s sensitivity and the number of objects detected (the degree of multiplexing). This combination is likely to enable a significant breakthrough in proteomics, particularly in the development of new drugs, clinical diagnostics, identification of molecular markers, and elucidation of the intracellular processes.     

Fluorescence data analysis on gel-based biochips

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.     

Photoimmobilization for microarrays

A photoimmobilization method has been developed for the preparation of microarray biochips. This photoimmobilization method makes it possible to easily covalently immobilize various types of organic molecules and cells on a chip. In addition, by using hydrophilic polymers as matrixes, it is possible to reduce nonspecific interactions with biological components. Various proteins, antibodies, and cells have been microarrayed using this technique, and interactions between these proteins, antibodies, and cells have been investigated. This type of microarray biochip will be important for academic applications such as genomics, proteomics, and cellomics, and clinical analyses.     

Immunological biochips for parallel detection of surface antigens and morphological analysis of cells

A biochip for detecting 26 cluster differentiation (CD), HLA-DR and IgM antigens on lymphocyte surface is described. The biochip, which represents a microarray of antibodies (IgG) against a panel of selected antigens immobilized on transparent plastic surfaces in 1.5-mm spots, was used for the study of normal and neoplastic lymphocytes and can also be used for determining percent of cells expressing definite surface antigens in lymphocyte suspensions. The results are consistent with data obtained by flow cytometry. The novel biochip technology entails a combination of conventional staining of cells immobilized on biochips and morphological analysis.     

生物芯片技术及其在基础生物科学研究中的应用

回顾了生物芯片的发展历史,重点介绍了生物芯片技术的两大技术基础：分子生物技术和微细加工生物技术;阐述了生物芯片技术的核心内容,总结了生物芯片的三大类型,并对生物芯片技术在生命科学基础研究中的应用进行了深入探讨和展望。     

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy

Near infrared spectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of near infrared spectroscopy from biochips are the basis for a novel method of fast, continuous, and contact‐free analysis of cell viability and real‐time monitoring of cell growth. The technique thus paves the way for a robust and reliable high‐throughput analysis of biochip‐embedded cell cultures.     

Optical Biochip with Multichannels for Detecting Biotin–Streptavidin Based on Localized Surface Plasmon Resonance

A rapid and accurate detection of molecular binding of antigen-antibody signaling in high throughput is of great importance for biosensing technology. We proposed a novel optical biochip with multichannels for the purpose of detection of biotin–streptavidin on the basis of localized surface plasmon resonance. The optical biochip was fabricated using photolithography to form the microarrays functioning with multichannels on glass substrate. There are different nanostructures in each microarray. Dry etching and nanosphere lithography techniques were applied to fabricate Ag nanostructures such as hemispheres, nanocylindricals, triangular, and rhombic nanostructures. We demonstrated that 100-nM target molecule (streptavidin) on these optical biochips can be easily detected by a UV-visible spectrometer. It indicated that period and shape of the nanostructures significantly affect the optical performance of the nanostructures with different shapes and geometrical parameters. Our experimental results demonstrated that the optical biochips with the multichannels can detect the target molecule using the microarrays structured with different shapes and periods simultaneously. Batch processing of immunoassay for different biomolecular through the different channels embedded on the same chip can be realized accordingly.     

几种新型生物芯片的研究进展

随着生物芯片技术的迅速发展,一些新型生物芯片,如生物电子芯片、凝胶元件微阵列芯片、药物控释芯片、毛细管电泳或层析芯片、PCR芯片及生物传感芯片等应运而生,这些芯片不同于常规的分子微阵列芯片,而是以各种结构微阵列为基础,用于分子杂交与扩增,以检测突变、分析多态性及测序,通过电泳及层析分离生物样品,控制药物释放以治疗疾病,作为生物传感器检测分子行为等,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域的新工具.     

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.     

Magnetoresistive-based biosensors and biochips

Over the past five years, magnetoelectronics has emerged as a promising new platform technology for biosensor and biochip development. The techniques are based on the detection of the magnetic fringe field of a magnetically labeled biomolecule interacting with a complementary biomolecule bound to a magnetic-field sensor. Magnetoresistive-based sensors, conventionally used as read heads in hard disk drives, have been used in combination with biologically functionalized magnetic labels to demonstrate the detection of molecular recognition. Real-world bio-applications are now being investigated, enabling tailored device design, based on sensor and label characteristics. This detection platform provides a robust, inexpensive sensing technique with high sensitivity and considerable scope for quantitative signal data, enabling magnetoresistive biochips to meet specific diagnostic needs that are not met by existing technologies.     

Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips

A method for fluorescently labeled single-stranded DNA (ssDNA) production during single-stage polymerase chain reaction (PCR) for subsequent hybridization on a biochip was described. This approach, whose efficiency was confirmed in the case of DARC gene, is considered as an alternative to two-stage nested PCR, consisting of two separate reactions: symmetric and asymmetric. Implementation of PCR in a single stage was achieved due to the use of a truncated excess primer in the second stage that does not anneal on the matrix during the cycles of symmetric stage of PCR and that enters the reaction after decrease of the annealing temperature in asymmetric stage. As a result, high efficiency of genotyping by means of hybridization on biochips is maintained. The suggested approach will allow us to reduce the time, working hours, and risk of contamination when researching biochips.     

Detection of biological macromolecules on a biochip dedicated to UV specific absorption

This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260 nm and 280 nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates. We analyze them through models and experiments and validate the possibility of measuring absorptions of the order of 10(-3). These multilayer structures display a high reflectivity, and maximize the interaction of the electric field with the biological element at the chip surface. Optimization of the experimental absorption, which includes effects such as roughness of the biochip, spectral and angular resolution of the optics, illumination, etc., is carried out with an inorganic ultraviolet absorber (titanium dioxide) deposit. We obtained an induced absorption contrast enhanced by a factor of 4.0, conferring enough sensitivity to detect monolayers of DNA or proteins. Experimental results on an Escherichia coli histidine-tagged methionyl-tRNA synthetase protein before and after complexation with an anti-polyHis specific antibody validate our biosensing technique. This label-free optical method may be helpful in controlling biochip coatings, and subsequent biological coupling at the surface of a biochip.     

Immunological biochips for studies of human erythrocytes

Immunological microarrays (biochips) for detecting erythrocyte surface antigens, viz., blood group antigens (A, B, 0) and Rhesus system antigens (D, E, e, C, and c), are described. The biochips represent transparent plastic supports onto which 1.5-mm spots of specific immobilized antibodies (IgM) are coated in different dilutions. The volume of tested blood samples is rather small (1–2 μl). Binding of erythrocytes to antibodies immobilized on the biochips is specific and allows further morphological analysis of bound cells. Analysis of the dynamics of cell detachment from biochip spots using a microfluidic chamber at different flow rates of the washing solution showed that combination of a biochip with a microfluidic chamber is a promising approach to concentration of cells of various immunotypes even if their content in the mixture is very low.     

Modeling of DNA hybridization kinetics for spatially resolved biochips

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.     

Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.     

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.