DDI-microFIA--A readily configurable microarray-fluorescence immunoassay based on DNA-directed immobilization of proteins

We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA-protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (microFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA-streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1A) in human blood serum samples. Detection limits down to 400 pg mL(-1) are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-microFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.     

GigaMatrix: an ultra high-throughput tool for accessing biodiversity

Recombinant approaches for tapping into the biodiversity present in nature for the discovery of novel enzymes and biosynthetic pathways can result in large gene libraries. Likewise, laboratory evolution techniques can result in large but potentially valuable libraries. Thorough screening of these libraries requires ultra high-throughput methods. The GigaMatrix^™ screening platform addresses this opportunity using reusable high-density plates with 100,000 to 1,000,000 through-hole wells in a microplate footprint. In addition to throughputs of over 10⁷ wells per day, the platform offers a significant reduction in reagent use and waste, has fully integrated automated “cherry picking,” and uses no complicated dispensing equipment. Wells containing putative hits from targeted fluorescent liquid phase assays are revealed by a fluorescent imaging system. Vision-guided robotics are utilized to recover hits by accessing individual 200 μm and smaller wells with a disposable sterile needle. The GigaMatrix platform has proven to be an effective and efficient tool for screening gene libraries for both discovery and evolution applications.     

基于元分析的差异表达基因识别

针对传统差异表达基因分析方法使用单一数据集,不能处理异质性特性数据集、分析结果偏差大的问题,提出了联合驱动排除的概念,设计实现一种使用多研究数据集的元分析算法。应用公共数据库GEO中GDS2490和GDS2491数据集对设计的算法与微芯片显著性分析方法进行了对比。实验结果表明:设计的算法可以对数据集实行联合驱动排除,与微芯片显著性分析方法相比,可以有效分析异质性特性的数据集,准确找到差异表达的基因,验证了算法的有效性。该方法为差异表达基因识别提供了新思路。     

Combinatorial Immunophenotyping of Cell Populations with an Electronic Antibody Microarray

Immunophenotyping is widely used to characterize cell populations in basic research and to diagnose diseases from surface biomarkers in the clinic. This process usually requires complex instruments such as flow cytometers or fluorescence microscopes, which are typically housed in centralized laboratories. Microfluidics are combined with an integrated electrical sensor network to create an antibody microarray for label‐free cell immunophenotyping against multiple antigens. The device works by fractionating the sample via capturing target subpopulations in an array of microfluidic chambers functionalized against different antigens and by electrically quantifying the cell capture statistics through a network of code‐multiplexed electrical sensors. Through a combinatorial arrangement of antibody sequences along different microfluidic paths, the device can measure the prevalence of different cell subpopulations in a sample from computational analysis of the electrical output signal. The device performance is characterized by analyzing heterogeneous samples of mixed tumor cell populations and then the technique is applied to determine leukocyte subpopulations in blood samples and the results are validated against complete blood cell count and flow cytometry results. Label‐free immunophenotyping of cell populations against multiple targets on a disposable electronic chip presents opportunities in global health and telemedicine applications for cell‐based diagnostics and health monitoring.     

A Boronic Acid Assay for the Detection of Mucin‐1 Glycoprotein from Cancer Cells

Cell surface glycoproteins are commonly aberrant in disease and act as biomarkers that facilitate diagnostics. Mucin‐1 (MUC1) is a prominent example, exhibiting truncated glycosylation in cancer. We present herein a boronic acid microplate assay for sensitive and high‐throughput detection of such glycoproteins. The immobilization of biotin–boronic acid 1 onto streptavidin plates generated a multivalent surface for glycoprotein recruitment and detection. We first validated the binding properties of 1 in solution through titrations with alizarin dye. Next, the microplate assay was explored through horseradish peroxidase (HRP) analysis as a proof‐of‐concept glycoprotein with chemiluminescence detection. Finally, this platform was applied for the detection of MUC1 directly from MCF‐7 human breast cancer cell lysates by using an HRP‐tagged antibody that targets the cancerous form of this glycoprotein. Sensitive, dose‐dependent detection of MUC1 was observed, showcasing the efficacy of this platform for detecting disease‐associated glycoproteins.     

Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface

A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.     

Quantification of Protein‐Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

The dynamic self‐organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro‐ and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self‐organization. Here, a new nanotechnology‐based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane‐remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label‐free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K _D) and kinetics (k_on and k_off). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1‐induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.     

Clinical proteomics: Promises,challenges and limitations of affinity arrays

After the establishment of DNA/RNA sequencing as a means of clinical diagnosis, the analysis of the proteome is next in line. As a matter of fact, proteome-based diagnostics is bound to be even more informative, since proteins are directly involved in the actual cellular processes that are responsible for disease. However, the structural variation and the biochemical differences between proteins, the much wider range in concentration and their spatial distribution as well as the fact that protein activity frequently relies on interaction increase the methodological complexity enormously, particularly if an accuracy and robustness is required that is sufficient for clinical utility. Here, we discuss the contribution that protein microarray formats could play towards proteome-based diagnostics.