Development of an innovative one-step nested PCR strategy for virus detection using the LNA technique

<正>Dear Editor,Polymerase chain reaction (PCR) is a molecular diagnostic technique that has been widely used for diagnosing viral diseases. Nested PCR (N-PCR) is a variation on the standard PCR technique that involves two amplification steps, yielding greater sensitivity and specificity. But there are two main disadvantages:the protocol is more complex than conven-     

A modified hybridization protocol for low-density diagnostic DNA microarrays

To promote the application of DNa microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42°C to 52°C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.     

DNA hybridization detection on electrical microarrays using coulostatic pulse technique

We demonstrated a novel application of transient coulostatic pulse technique for the detection of label free DNA hybridization on nm-sized gold interdigitated ultramicroelectrode arrays (Au-IDA) made in silicon technology. The array consists of eight different positions with an Au-IDA pair at each position arranged on the Si-based Biochip. Immobilization of capture probes onto the Au-IDA was accomplished by self-assembling of thiol-modified oligonucleotides. Target hybridization was indicated by a change in the magnitude of the time dependant potential relaxation curve in presence of electroactive Fe(CN)(6)(3-) in the phosphate buffer solution. While complementary DNA hybridization showed 50% increase in the relaxation potential, the non-complementary DNA showed a negligible change. A constant behaviour was noted for all positions. The dsDNA specific intercalating molecule, methylene blue, was found to be enhancing the discrimination effect. The changes in the relaxation potential curves were further corroborated following the ELISA like experiments using ExtraAvidine alkaline phosphatase labelling and redox recycling of para-aminophenol phosphate at IDAs. The coulostatic pulse technique was shown to be useful for identifying DNA sequences from brain tumour gene CK20, human herpes simplex virus, cytomegalovirus, Epstein-Barr virus and M13 phage. Compared to the hybridization of short chain ONTs (27 mers), the hybridization of long chain M13 phage DNA showed three times higher increase in the relaxation curves. The method is fast enough to monitor hybridization interactions in milli or microsecond time scales and is well suitable for miniaturization and integration compared to the common impedance techniques for developing capacitative DNA sensors.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

Label-free detection methods for protein microarrays

With the growth of the "-omics" such as functional genomics and proteomics, one of the foremost challenges in biotechnologies has become the development of novel methods to monitor biological process and acquire the information of biomolecular interactions in a systematic manner. To fully understand the roles of newly discovered genes or proteins, it is necessary to elucidate the functions of these molecules in their interaction network. Microarray technology is becoming the method of choice for such a task. Although protein microarray can provide a high throughput analytical platform for protein profiling and protein-protein interaction, most of the current reports are limited to labeled detection using fluorescence or radioisotope techniques. These limitations deflate the potential of the method and prevent the technology from being adapted in a broader range of proteomics applications. In recent years, label-free analytical approaches have gone through intensified development and have been coupled successfully with protein microarray. In many examples of label-free study, the microarray has not only offered the high throughput detection in real time, but also provided kinetics information as well as in situ identification. This article reviews the most significant label-free detection methods for microarray technology, including surface plasmon resonance imaging, atomic force microscope, electrochemical impedance spectroscopy and MS and their applications in proteomics research.     

Silk as an innovative biomaterial for cancer therapy

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.     

Bioflocculation as an innovative harvesting strategy for microalgae

Microalgae are a promising new source of biomass for the production of third generation biofuels but, so far, the majority of microalgal biomass has been used for high-value applications. New low-cost technologies are needed to make the production and processing of microalgae economically feasible for low-value applications. A major challenge lies in the harvesting of microalgae, which requires a cost-efficient separation technology. Flocculation, especially bioflocculation, is an attractive low-cost separation technology. Various new bioflocculation strategies have been claimed to generate major advances in cost-efficient harvesting. Here, we review the recent advances in bioflocculation based on algal–bacterial, algal–fungal, or algal–algal interactions within the framework of microalgae biomass harvesting for biofuel production. We also discuss recent advances using infochemicals and genetic engineering for the induction of bioflocculation.     

Detection of CFTR mutations using ARMS and low-density microarrays

The amplification refractory mutation system (ARMS) is routinely used for the identification of specific mutations within genomes. This PCR-based assay, although simple, is performed at a low-throughput scale, usually requiring gel-electrophoresis for the identification of specific mutations. We have applied the ARMS technology to a low-density microarray system to facilitate the needs of the medical clinic; high-throughput capabilities and ease-of-use. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene (DeltaF508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Amine-modified probes specific for both the wild-type and mutant forms of each mutation site were attached to glass substrates. Following hybridization of the PCR fragments to the attached probes (in a low-density microarray format), confirmation of the presence of specific sequences was achieved using a commercial scanner, as well as a fabricated low-cost fluorescent detector and applicable software. The novel combination of the ARMS and low-density microarray technologies allows for a high-throughput, simple means to rapidly identify multiple known mutations for many genetic diseases including cystic fibrosis.     

Enhanced detection of autoantibodies on protein microarrays using a modified protein digestion technique

High-throughput studies to determine differential immune (humoral) response to diseases are becoming of increasing interest because the information they provide can help in early diagnosis as well as monitoring of therapeutics. Protein microarrays are a high-throughput and convenient technology that can be applied to the study of the humoral response. Proteins can be arrayed on slides and then probed with serum from different classes of patients to observe differences that may exist among autoantibodies that reflect differences in disease states. However, such studies may be difficult to interpret due to the weak overall signal response of such protein microarrays. We propose that this weak signal response is due to the physical positioning of the disease proteins that renders them sterically hindered from binding partners in the serum. In this study, we hypothesize that reducing the complexity and size of the disease proteins by chemical digestion using cyanogen bromide (CNBr) may enhance the overall signal from the humoral response and facilitate visualization of disease-specific responses in various classes of serum. A modified protein microarray methodology using CNBr digestion is presented here. The new workflow was applied to a set of 10 serum samples from healthy subjects, 10 from patients with chronic pancreatitis and 10 from patients diagnosed with pancreatic cancer and the results were compared to results obtained in the absence of CNBr digestion. CNBr digestion allowed the identification of 10 additional autoantibodies that responded to serum, 5 of which were unique to pancreatitis and cancer sera. This new methodology may increase the sensitivity of microarray studies measuring autoantibodies in serum.     

Aptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates

This study presents and discusses the application of Cy3‐labeled aptamers (where Cy3 is indocarbocyanine) directed against the his‐tag (where his is histidine) for the detection of his‐tagged proteins on microarrays in a so‐called reverse phase assay. These types of assays are widely used tools in protein microarray technology. Up to now antibodies are usually applied as detection molecules. Here, two different spotting techniques, contact and noncontact spotting, as well as different types of slides, aldehyde‐modified glass slides and nitrocellulose membrane coated slides, were examined and compared. Through this study, we validated the importance of a high protein‐binding capacity of the microarray, and the labeling position of the fluorophore within the aptamer. Purified his‐tagged PFEI (Pseudomonas fluorescence esterase I) was used as a model system. Concentrations of PFEI‐his as low as 30 nM were detectable. These results demonstrate the applicability of aptamers as stable detection molecules in protein assays. Additionally, the reverse phase assay was found to be suitable for the detection of PFEI‐his in cell lysates. This might be of further interest in monitoring of protein production and purification processes.     

Small-molecule microarrays: development of novel linkers and an efficient detection method for bound proteins

Novel isocyanate and diazoketone linkers possessing polyoxypropylenediamine as a spacer for small-molecule microrray are developed. White light interferometry is introduced to detect bound proteins on the glass slides without using chemically modified proteins.     

Self-self hybridization as an alternative experiment design to dye swap for two-color microarrays

Dye-specific bias effects, commonly observed in the two-color microarray platform, are normally corrected using the dye swap design. This design, however, is relatively expensive and labor-intensive. We propose a self-self hybridization design as an alternative to the dye swap design. In this design, the treated and control samples are labeled with Cy5 and Cy3 (or Cy3 and Cy5), respectively, without dye swap, along with a set of self-self hybridizations on the control sample. We compare this design with the dye swap design through investigation of mouse primary hepatocytes treated with three peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists at three dose levels. Using Agilent's Whole Mouse Genome microarray, differentially expressed genes (DEG) were determined for both the self-self hybridization and dye swap designs. The DEG concordance between the two designs was over 80% across each dose treatment and chemical. Furthermore, 90% of DEG-associated biological pathways were in common between the designs, indicating that biological interpretations would be consistent. The reduced labor and expense for the self-self hybridization design make it an efficient substitute for the dye swap design. For example, in larger toxicogenomic studies, only about half the chips are required for the self-self hybridization design compared to that needed in the dye swap design.     

Peptide-protein microarrays for the simultaneous detection of pathogen infections

We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.     

Protein microarrays as tools for functional proteomics

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.     

Biomolecule-embedded metal-organic frameworks as an innovative sensing platform

Technological advancements combined with materials research have led to the generation of enormous types of novel substrates and materials for use in various biological/medical, energy, and environmental applications. Lately, the embedding of biomolecules in novel and/or advanced materials (e.g., metal-organic frameworks (MOFs), nanoparticles, hydrogels, graphene, and their hybrid composites) has become a vital research area in the construction of an innovative platform for various applications including sensors (or biosensors), biofuel cells, and bioelectronic devices. Due to the intriguing properties of MOFs (e.g., framework architecture, topology, and optical properties), they have contributed considerably to recent progresses in enzymatic catalysis, antibody-antigen interactions, or many other related approaches. Here, we aim to describe the different strategies for the design and synthesis of diverse biomolecule-embedded MOFs for various sensing (e.g., optical, electrochemical, biological, and miscellaneous) techniques. Additionally, the benefits and future prospective of MOFs-based biomolecular immobilization as an innovative sensing platform are discussed along with the evaluation on their performance to seek for further development in this emerging research area.     

Protein microarrays and novel detection platforms

The field of proteomics has undergone rapid advancements over the last decade and protein microarrays have emerged as a promising technological platform for the challenging task of studying complex proteomes. This gel-free approach has found an increasing number of applications due to its ability to rapidly and efficiently study thousands of proteins simultaneously. Different protein microarrays, including capture arrays, reverse-phase arrays, tissue microarrays, lectin microarrays and cell-free expression microarrays, have emerged, which have demonstrated numerous applications for proteomics studies including biomarker discovery, protein interaction studies, enzyme-substrate profiling, immunological profiling and vaccine development, among many others. The need to detect extremely low-abundance proteins in complex mixtures has provided motivation for the development of sensitive, real-time and multiplexed detection platforms. Conventional label-based approaches like fluorescence, chemiluminescence and use of radioactive isotopes have witnessed substantial advancements, with techniques like quantum dots, gold nanoparticles, dye-doped nanoparticles and several bead-based methods now being employed for protein microarray studies. In order to overcome the limitations posed by label-based technologies, several label-free approaches like surface plasmon resonance, carbon nanotubes and nanowires, and microcantilevers, among others, have also advanced in recent years, and these methods detect the query molecule itself. The scope of this article is to outline the protein microarray techniques that are currently being used for analytical and function-based proteomics and to provide a detailed analysis of the key technological advances and applications of various detection systems that are commonly used with microarrays.     

Colorimetric silver detection of DNA microarrays

Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method. Microarrays are currently detected in fluorescence using a sophisticated confocal laser-based scanner. In this work, we present a new colorimetric detection method which is intented to make the use of microarray a powerful procedure and a low-cost tool in research and clinical settings. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotinylated DNA. This colorimetric method has been compared to the Cy-3 fluorescence method. The detection limit of both methods was equivalent and corresponds to 1 amol of biotinylated DNA attached on an array. Scanning and data analysis of the array were obtained with a colorimetric-based workstation.