Study on The Method of Quantitative Analysis of Serum Ferritin and Soluble Transferrin Receptor with Protein Microarray Technology

Objective To establish and evaluate a protein serum ferritin （SF） and soluble transferrin receptor microarray method for combined measurement of （sTfR）. Methods Microarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies Ill. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples. Results By comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR. Conclusion The present study has established a protein microarray method for combined measurement of SF and sTfR.

Protein Microarrays for Quantitative Detection of PAI-1 in Serum

Objective： Plasminogen activator inhibitor-1 （PAI-1）, one crucial component of the plasminogen activator system, is a major player in the pathogenesis of many vascular diseases as well as in cancer. High levels of PAI-1 in breast cancer tissue are associated with poor prognosis. The aim of this study is to evaluate rigorously the potential of serum PAI-1 concentration functioning as a general screening test in diagnostic or prognostic assays. Methods： A protein-microarray-based sandwich fluorescence immunoassay （FIA） was developed to detect PAI-1 in serum. Several conditions of this microarray-based FIA were optimized to establish an efficacious method. Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray. Results： The median serum PAI-1 level of breast cancer patients was higher than that of healthy women （109.7 ng/ml vs. 63.4 ng/ml）. Analysis of covariance revealed that PAI-1 levels of the two groups were significantly different （P0.001） when controlling for an age effect on PAI-1 levels. However, PAI-1 values in TNM stage I？IV patients respectively were not significantly different from each other. Conclusion： This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAI-1.

Enhancing the quality metric of protein microarray image

The novel method of improving the quality metric of protein microarray image presented in this paper reduces impulse noise by using an adaptive median filter that employs the switching scheme based on local statistics characters; and achieves the impulse detection by using the difference between the standard deviation of the pixels within the filter window and the current pixel of concern. It also uses a top-hat filter to correct the background variation. In order to decrease time consumption, the top-hat filter core is cross structure. The experimental results showed that, for a protein microarray image contaminated by impulse noise and with slow background variation, the new method can significantly increase the signal-to-noise ratio, correct the trends in the background, and enhance the flatness of the background and the consistency of the signal intensity.

Improving the sensitivity of protein microarray by evanescent-field-induced fluorescence

To improve the sensitivity of protein microarray, a prism surface replaces the surface of the common microscope slide.The protein targets arrayed on the surface are hybridized and labelled by fluorescent probes. Evanescent excitation occurs when the convergent laser reaches the surface, and a photomultiplier tube detects the emitted fluorescent signal. A two-dimensional actuator scans the whole surface to achieve planar laser excitation and fluorescence collection. The penetration depth of the evanescent field into the protein targets is only some hundred nanometers and can be controlled by different incident angle of the laser beam, so the undesired background signals are reduced dramatically and the detection sensitivity is improved by a factor of 50 to 100 comparing to confocal excitation. This approach can detect low abundance analytes without signal amplification.

LABEL-FREE DETECTION OF PROTEIN MICROARRAY WITH HIGH THROUGHPUT SURFACE PLASMON RESONANCE IMAGING(SPRI)

A surface plasmon resonance imaging(SPRI)system was developed for the discrimination of proteins on a gold surface.As a label-free and high-throughput technique,SPRI enables simultaneously monitoring of the biomolecular interactions at low concentrations.We used SPRI as a label-free and parallel method to detect different proteins based on protein microarray.Bovine Serum Albumin(BSA),Casein and Immunoglobulin G(IgG)were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6×6 matrix.These proteins can be discriminated directly by changing the incident angle of light.Excellent reproducibility for label-free detection of protein molecules was achieved.This SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.

Detection of hybridization of protein microarrays using an oblique-incidence reflectivity difference method

Mouse-Immunoglobulin G(mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference(OI-RD) method.Two detection signals,consisting of an imaginary part(Im{Δp-Δs}) and a real part(Re{Δp-Δs}) of OI-RD,were obtained simultaneously.The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans.In particular,we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates.The detection sensitivity of OI-RD can reach 14 fg.These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.

Label-free detection repeatability of protein microarrays by oblique-incidence reflectivity difference method

We examine the repeatabilities of oblique-incidence reflectivity difference(OIRD) method for label-free detecting biological molecular interaction using protein microarrays.The experimental results show that the repeatabilities are the same in a given microarray or microarray-microarray and are consistent,indicating that OIRD is a promising label-free detection technique for biological microarrays.

Functional protein microarray： an ideal platform for investigating protein binding property

Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements have also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among these applications, detection of protein binding properties, such as protein-protein interactions （PPIs）, protein-DNA interactions （PDIs）, protein-RNA interactions, and antigen-antibody interactions, are straightforward and have substantial impacts on many research fields. In this review, we will focus on the recent progresses in protein-protein, protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions. We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that protein microarrays will soon become an indispensible tool for both basic research and clinical applications.

Protein microarray biosensors based on imaging ellipsometry techniques and their applications

After years of development,biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems.Their experimental setups,detection principles,and biological and clinical applications are briefly reviewed.

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Pro?ling

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens （DIRAGs） could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer （CRC） patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2and mTOR signaling. Ribosomal proteins （e.g., RPL7, RPL22, and RPL27A） and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, dif- ferential pathways were observed between the CRC and control samples. Furthermore, 103 DIR- AGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 ＂CRC genes.＂ These data indicate that immunomies profiling on protein mieroarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

Serological profiling of Crohn’s disease and ulcerative colitis patients reveals anti-microbial antibody signatures

BACKGROUND The healthcare burden of inflammatory bowel disease(IBD)is rising globally and there are limited non-invasive biomarkers for accurate and early diagnosis.AIM To understand the important role that intestinal microbiota play in IBD pathogenesis and identify anti-microbial antibody signatures that benefit clinical management of IBD patients.METHODS We performed serological profiling of 100 Crohn’s disease(CD)patients,100 ulcerative colitis(UC)patients and 100 healthy controls against 1173 bacterial and 397 viral proteins from 50 bacteria and 33 viruses on protein microarrays.The study subjects were randomly divided into discovery(n=150)and validation(n=150)sets.Statistical analysis was performed using R packages.RESULTS Anti-bacterial antibody responses showed greater differential prevalence among the three subject groups than anti-viral antibody responses.We identified novel antibodies against the antigens of Bacteroidetes vulgatus(BVU_0562)and Streptococcus pneumoniae(SP_1992)showing higher prevalence in CD patients relative to healthy controls.We also identified antibodies against the antigen of Streptococcus pyogenes(SPy_2009)showing higher prevalence in healthy controls relative to UC patients.Using these novel antibodies,we built biomarker panels with area under the curve(AUC)of 0.81,0.87,and 0.82 distinguishing CD vs control,UC vs control,and CD vs UC,respectively.Subgroup analysis revealed that penetrating CD behavior,colonic CD location,CD patients with a history of surgery,and extensive UC exhibited highest antibody prevalence among all patients.We demonstrated that autoantibodies and anti-microbial antibodies in CD patients had minimal correlation.CONCLUSION We have identified antibody signatures for CD and UC using a comprehensive analysis of antimicrobial antibody response in IBD.These antibodies and the source microorganisms of their target antigens improve our understanding of the role of specific microorganisms in IBD pathogenesis and,after future validation,should aid early and accurate diagnosis of IBD.

Phase-sensitive plasmonic biosensor using a portable and large field-of-view interferometric microarray imager

Nanophotonics,and more specifically plasmonics,provides a rich toolbox for biomolecular sensing,since the engineered metasurfaces can enhance light–matter interactions to unprecedented levels.So far,biosensing associated with high-quality factor plasmonic resonances has almost exclusively relied on detection of spectral shifts and their associated intensity changes.However,the phase response of the plasmonic resonances have rarely been exploited,mainly because this requires a more sophisticated optical arrangement.Here we present a new phase-sensitive platform for high-throughput and label-free biosensing enhanced by plasmonics.It employs specifically designed Au nanohole arrays and a large field-of-view interferometric lens-free imaging reader operating in a collinear optical path configuration.This unique combination allows the detection of atomically thin(angstrom-level)topographical features over large areas,enabling simultaneous reading of thousands of microarray elements.As the plasmonic chips are fabricated using scalable techniques and the imaging reader is built with low-cost off-the-shelf consumer electronic and optical components,the proposed platform is ideal for point-of-care ultrasensitive biomarker detection from small sample volumes.Our research opens new horizons for on-site disease diagnostics and remote health monitoring.

内嵌钆金属富勒烯通过抑制氧化还原介导的内皮细胞迁移诱导肿瘤血管正常化并增强化疗疗效

肿瘤血管正常化(TVN)可以通过逆转异常的肿瘤血管,从而增强抗癌效率,特别是增加药物的瘤内输送.内皮细胞在血管生成中起着至关重要的作用,但持续调控内皮细胞迁移以改善TVN是巧妙而具有挑战性的.本文提出了一种基于具有纳米尺寸和抗氧化能力的富勒烯纳米颗粒(FNPs)抑制内皮细胞迁移的可能策略来实现TVN.本研究证明FNPs在体外通过其抗氧化作用抑制细胞迁移.丙氨酸修饰的钆富勒烯(GFA)表现出优异的TVN效果,并在体内抑制肿瘤生长.在蛋白质微阵列的帮助下,确认GFA在机制上是通过抑制粘着斑通路,从而抑制内皮细胞的迁移.随后,得益于GFA的血管正常化效应,显著增强了化疗疗效.总之,本文证明了富勒烯纳米材料在肿瘤血管正常化中的应用潜力,并拓展了其在癌症纳米医学材料设计中应用的可能性.

Study roadmap for high-throughput development of easy to use and affordable biomarkers as diagnostics for tropical diseases:a focus on malaria and schistosomiasis

Background:Interventions are currently being used against‘infectious diseases of poverty’,which remain highly debilitating and deadly in most endemic countries,especially malaria,schistosomiasis,echinococcosis and African sleeping sickness.However,major limitations of current‘traditional’methods for diagnosis are neither simple nor convenient for population surveillance,and showed low sensitivity and specificity.Access to novel technologies for the development of adequate and reliable tools are expressly needed.A collaborative project between African Network for Drugs and Diagnostics Innovation and partner institutions in Africa and China aims to screen suitable serological biomarkers for diagnostic pipelines against these‘diseases of the poor’.Methods:Parasite-specific exposed versus unexposed individuals were screened and sera or urine/stools were collected through case-control studies in China and African countries.Target genes/open reading frames were selected,then will be cloned and cell-free expressed,quantified and immuno-detected.Target antigens/epitopes will be probed and screened with sera from exposed or unexposed individuals using a high-throughput antigen screening platform as the study progresses.The specificity and sensitivity of highly immunoreactive biomarkers will be evaluated as well,using enzyme-linked immunosorbent assays or dipsticks.Discussion:This roadmap explicitly unfolds the integrated operating procedures with focus on malaria and schistosomiasis,for the identification of suitable biomarkers that will aid the prioritization of diagnostics for population use.However,there is need to further validate any new diagnostic through comparison with standard methods in field deployable tests for each region.Our expectations for the future are to seek regulatory approval and promote the use of diagnostics in endemic areas.

COVID-ONE-hi:The One-stop Database for COVID-19-specific Humoral Immunity and Clinical Parameters

Coronavirus disease 2019(COVID-19),which is caused by SARS-CoV-2,varies with regard to symptoms and mortality rates among populations.Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19.However,differences in immune responses and clinical features among COVID-19 patients remain largely unknown.Here,we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters(named COVID-ONE-hi).COVID-ONE-hi is based on the data that contain the IgG/IgM responses to 24 full-length/truncated proteins corresponding to 20 of 28 known SARS-CoV-2 proteins and 199 spike protein peptides against 2360 serum samples collected from 783 COVID-19 patients.In addition,96 clinical parameters for the 2360 serum samples and basic information for the 783 patients are integrated into the database.Furthermore,COVID-ONE-hi provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups.A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters.After the“START”button is clicked,one can readily obtain a comprehensive analysis report for further interpretation.COVID-ONE-hi is freely available at www.COVID-ONE.cn.