Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis

Background

Since the discovery of human immunodeficiency virus (HIV-1) twenty years ago, AIDS has become one of the most studied diseases. A number of viruses have subsequently been identified to contribute to the pathogenesis of HIV and its opportunistic infections and cancers. Therefore, a multi-virus array containing eight human viruses implicated in AIDS pathogenesis was developed and its efficacy in various applications was characterized.

Results

The amplified open reading frames (ORFs) of human immunodeficiency virus type 1, human T cell leukemia virus types 1 and 2, hepatitis C virus, Epstein-Barr virus, human herpesvirus 6A and 6B, and Kaposi's sarcoma-associated herpesvirus were spotted on glass slides and hybridized to DNA and RNA samples. Using a random priming method for labeling genomic DNA or cDNA probes, we show specific detection of genomic viral DNA from cells infected with the human herpesviruses, and effectively demonstrate the inhibitory effects of a cellular cyclin dependent kinase inhibitor on viral gene expression in HIV-1 and KSHV latently infected cells. In addition, we coupled chromatin immunoprecipitation with the virus chip (ChIP-chip) to study cellular protein and DNA binding.

Conclusions

An amplicon based virus chip representing eight human viruses was successfully used to identify each virus with little cross hybridization. Furthermore, the identity of both viruses was correctly determined in co-infected cells. The utility of the virus chip was demonstrated by a variety of expression studies. Additionally, this is the first demonstrated use of ChIP-chip analysis to show specific binding of proteins to viral DNA, which, importantly, did not require further amplification for detection.     

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.     

Preparation of a visual protein chip for detection of IgG against Treponema pallidum

A rapid, visual protein chip method for the detection of IgG against Treponema pallidum was developed using Staphylococcus protein A-modified gold nanoparticles. After recombinant T. pallidum antigen Tpp47 was arrayed on aldehyde-coated slides, qualitative and semiquantitative assay results were obtained from the hybridization signal and the subsequent gray stain values. The Tpp47-specific serum antibody was bound to immobilized Tpp47 antigen on the functionalized glass slides. The Staphylococcus protein A-modified gold nanoparticle probe exclusively recognized anti-Tpp47 IgG. The "sandwich" format hybridization signal was then amplified with silver staining, a high sensitivity visual detection technique. It took 3.5 h to prepare the detection protein chip, but only 20 min for real sample detection. The lowest titer of detectable antibody for this method was 1:128, which correlates to approx 1 ng/mL. Furthermore, the results can only be seen unaided, which drastically reduces the cost of detection, but can also be kept for a long time. The data also confirmed the proposed method's specificity, sensitivity, and convenience. As a result, the method could be applied as an alternative diagnostic tool in the clinical diagnosis of T. pallidum.     

Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.     

Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance

We have developed a biomimetic sensor for the detection of human immunodeficiency virus type 1 (HIV-1) related protein (glycoprotein 41, gp41) based on epitope imprinting technique. gp41 is the transmembrane protein of HIV-1 and plays an important role in membrane fusion between viruses and infected cells. It is an important index for determining the extent of HIV-1 disease progression and the efficacy of therapeutic intervention. In this work, dopamine was used as the functional monomer and polymerized on the surface of quartz crystal microbalance (QCM) chip in the presence of template, a synthetic peptide with 35 amino acid residues, analogous to residues 579-613 of the gp41. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the QCM chip. QCM measurement showed that the resulting MIP film not only had a great affinity towards the template peptide, but also could bind the corresponding gp41 protein specifically. The dissociation constant (K(d)) of MIP for the template peptide was calculated to be 3.17 nM through Scatchard analysis, which was similar to those of monoclonal antibodies. Direct detection of the gp41 was achieved quantitatively using the resulting MIP-based biomimetic sensor. The detection limit of gp41 was 2 ng/mL, which was comparable to the reported ELISA method. In addition, the practical analytical performance of the sensor was examined by evaluating the detection of gp41 in human urine samples with satisfactory results.     

On a chip

The future of clinical and POC BioMEMS is very bright. With an increasing emphasis on the personalization of medicine and the rising costs of health care, early detection and diagnostics at the POC will be even more important. Early detection implies early intervention, resulting in the saving of lives and reducing overall spending. The potential impact of these technologies on the early diagnosis and management of both communicable and noncommunicable diseases is very high. Many grand challenges applications are possible, e.g., routine tests such as complete blood cell count on a chip that an individual can perform at home; detection of cardiac markers from blood after a perceived heart attack; detection of cancer markers such as exosomes, CTCs from blood, or protein biomarkers in serum; and detection of infectious agents such as virus and bacteria for public health. These applications are expected to result in new diagnostic assays for home, doctor's office, clinical laboratories, and various POC settings.     

针对多种强致病性病毒的基因芯片检测方法的建立

为了制备灵敏的可检测多种烈性病毒性病原体的基因芯片,本研究设计了针对21种烈性病毒性病原体的基因芯片检测探针,每种5条,长50 bp.并以甲病毒属的基孔肯亚病毒和黄病毒属的黄热病毒细胞培养物为检测模型,摸索了合适的病毒基因处理与扩增方法.将提取的病毒RNA先用DNase Ⅰ处理,以去除掉其中的DNA分子,然后利用病毒属特异性引物进行反转录,以引导病毒基因组的合成,从而尽可能地减少宿主细胞基因成分的干扰.进行随机PCR扩增后将扩增产物与基因芯片进行杂交,分别出现了4条基孔肯亚病毒探针信号和5条黄热病毒的探针信号,说明所设计的检测探针具有较好的特异性,可用于这2种病毒的特异性检测.这种病毒基因样品的处理和扩增方法也为此基因芯片的临床应用奠定了基础.     

Development of protein microarray technology to monitor biomarkers of rheumatoid arthritis disease

Most biological processes are mediated by complex networks of molecular interactions involving proteins. The analysis of protein expression in biological samples is especially important in the identification and monitoring of biomarkers for disease progression and therapeutic endpoints. In this paper, the development of a protein microarray format for multiplexed quantitative analysis of several potential markers for rheumatoid arthritis (RA) is described. Development of a high-performance protein microarray system depends on several key parameters such as surface chemistry, capture agents, immobilization technology, and methods used for signal detection and quantification. Several technical possibilities were investigated and compared: poly-L-lysine versus self-assembled monolayer of octadecyl phosphoric acid ester for surface chemistries; noncontact piezoelectric versus contact printing technology for antibody deposition; CCD camera capture versus fluorescent scanning for image detection; and the concentration of coating antibody. On the basis of reproducibility, signal-to-noise ratio, and sensitivity we have selected self-assembled monolayer, noncontact piezoelectric printer, and high-read-out fluorescence scanning for our microarray format. This format was used to perform multiplexed quantitative analysis of several potential markers of disease progression of rheumatoid arthritis: IL-1, IL-6, IL-8, MCP-1, and SAA. Some assays, such as MCP-1, provided a working range that covered physiologically relevant concentrations. Other assays, such as IL-6 and SAA, lacked sensitivity or were too sensitive for measuring biological concentrations, respectively. The results described demonstrate the applicability of protein microarrays to monitor RA markers; however, sandwich assay methodologies need to be further optimized to measure the appropriate biological ranges of these markers on one chip.     

High-throughput multi-antigen microfluidic fluorescence immunoassays

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.     

四种方法检测乙型肝炎病毒基因型的比较

目的比较四种方法检测乙型肝炎病毒基因型的差异性。方法对36例乙型肝炎患者的血清分别采用测序技术,荧光定量PCR技术,恒温扩增技术,基因芯片技术进行乙肝病毒基因型的检测。结果36份血清以测序技术为金标,观测荧光定量PCR技术特异性100%,敏感性83%,恒温扩增技术特异性100%,敏感性89%,基因芯片技术特异性100%,敏感度92%。结论目前临床采用的四种方法检测乙型肝炎病毒基因型的特异性相同,敏感度以测序为金标准,基因芯片技术最灵敏,其次为恒温扩增技术,最后为荧光定量PCR技术。     

用SARS冠状病毒全基因组芯片杂交方法分析SARS-CoV

为从临床样品中检测和分析SARSCoV病毒打基础,并为分析SARSCoV病毒的复制和转录等机理提供一种有效方法。以SARS冠状病毒TOR2株序列作为标准设计和制备一种覆盖SARS冠状病毒全基因组的寡聚核苷酸芯片,探针长度为70nt,每相邻的探针序列重复25nt,共660条。用该芯片分析了细胞培养的SARSCoV病毒总RNA、7个SARSCoV病毒的基因克隆片段。对RNA样品用随机引物进行反转录PCR获得cDNA。对DNA用随机引物扩增和dUTPcy3标记。结果用这种芯片杂交检测SARSCoV病毒RNA可见阳性信号呈全基因组分布,并且有多处连续的阳性信号点;用正常人的白细胞RNA为对照,杂交未出现明显阳性信号。检测7个SARSCoV病毒基因克隆片段,在该片段相应的探针区段出现连续阳性信号点。这种方法可有效地检测和分析样品中SARS冠状病毒全基因组的信息。     

The fabrication of protein chip based on surface plasmon resonance for detection of pathogens

Protein chip based on surface plasmon resonance (SPR) was developed for detection of pathogens existing in contaminated environment, such as Escherichia coli O157:H7, Salmonella typhimurium, Legionella pneumophila, and Yersinia enterocolitica. Protein G was immobilized to endow the orientation of antibody molecules on the SPR surface. The pathogen binding of the protein chip was investigated by SPR spectroscopy. Consequently, it was found that the four kinds of pathogen could be selectively detected by using SPR-based protein chip.     

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence

A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nano-assembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.     

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.     

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Automated 10-channel capillary chip immunodetector for biological agents detection

The automated 10-channel capillary chip immunodetector (10K-IDWG) is a prototype, which has been developed for automatically operated biological agents (BA) point detection. The current technology uses a chemiluminescence capillary immunoassay (EIA) technique in combination with integrated microfluidics and allows the highly sensitive and rapid detection and preliminary identification of multiple BA in aqueous solutions in the laboratory. The chemiluminescence capillary EIA are performed within a disposable capillary chip containing 10 fused-silica capillaries arranged in parallel coated with selected capture antibodies. A multianode-photomultiplier array is used to detect chemiluminescence intensity in each capillary. Reservoirs for reagents and buffers and a waste disposal reservoir are integrated. This paper describes the technology of the 10K-IDWG and its evaluation with three different BA, the toxin staphylococcal enterotoxin B (SEB), the bacterial analyte Escherichia coli (E. coli) O157:H7 as a model for bacterial pathogens, and the bacteriophage M13 as a model for virus pathogens. The 10K-IDWG is able to detect the above mentioned three BA in an aqueous sample within 29 min (single analyte-detection and multiplexing). Limits of detection (LOD) are 0.1 ng/ml for SEB, 10(4)cfu/ml for E. coli O157:H7, and 5x10(5) pfu/ml for M13. Cross reactivities between the three assays were not observed.     

An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays

An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip

In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.