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.

Label-Free and Real-Time Monitor of Binding and Dissociation Processes between Protein A and Swine IgG by Oblique-Incidence Reflectivity Difference Method

Life science has a need for detection methods that are label-free and real-time. In this paper, we have selected staphylococcal protein A （SPA） and swine immunoglobulin G （IgG）, and monitor the bindings between SPA and swine IgG with different concentrations, as well as the dissociations of SPA-swine IgG complex in different pH values of phosphate buffer by oblique-incidence reflectivity difference （OIRD） in a label-free and real-time fashion. We obtain the ON and OFF reaction dynamic curves corresponding to the bindings and dissociations of SPA and swine IgG. Through our analysis of the experimental results, we have been able to obtain the damping coefficients and the dissociation time of SPA and swine IgG for different pH values of the phosphate buffer. The results prove that the OIRD technique is a competing method for monitoring the dynamic processes of biomolecule interaction and achieving the quantitative information of reaction kinetics.

Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.

Circulating proteomic biomarkers for diagnosing sporadic amyotrophic lateral sclerosis:a cross-sectional study

Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.

低钾胁迫下高钾烤烟苗期根系差异表达蛋白分析

为了解高钾烤烟苗期根系在低钾胁迫下的差异表达蛋白及其作用机理,本研究分别提取低钾和正常钾营养液培育的高钾烤烟品系HKDN-5的苗期根系蛋白,酶解后采用液相色谱串联质谱和label-free蛋白定量技术对差异表达蛋白进行鉴定分析。结果表明,与正常钾相比,低钾组共发现681个差异表达蛋白,其中有359个发生下调表达,322个上调表达。差异表达蛋白主要参与脂质代谢、次生代谢、碳水化合物代谢和遗传信息加工等途径。其中,下调蛋白具有翻译后修饰、蛋白质转换、伴侣蛋白相关功能的数量最多,上调蛋白主要为一般功能相关蛋白。差异倍数较大的蛋白主要与代谢和应激反应过程相关。以上结果表明,低钾胁迫产生的差异蛋白主要参与物质代谢及应激反应等相关过程。

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.

Detection of protein microarrays by oblique-incidence reflectivity difference technique

Biological microarrays with different proteins and different protein concentrations are detected without external labeling by an oblique-incidence reflectivity difference (OIRD) technique. The initial experiment results reveal that the intensities of OIRD signals can distinguish the different proteins and concentrations of protein. The OIRD technique promises feasible applications to life sciences for label-free and high-throughput detection.

Nanoplasmonic mid-infrared biosensor for in vitro protein secondary structure detection

Plasmonic nanoantennas offer new applications in mid-infrared(mid-IR)absorption spectroscopy with ultrasensitive detection of structural signatures of biomolecules,such as proteins,due to their strong resonant near-fields.The amide I fingerprint of a protein contains conformational information that is greatly important for understanding its function in health and disease.Here,we introduce a non-invasive,label-free mid-IR nanoantenna-array sensor for secondary structure identification of nanometer-thin protein layers in aqueous solution by resolving the content of plasmonically enhanced amide I signatures.We successfully detect random coil to crossβ-sheet conformational changes associated withα-synuclein protein aggregation,a detrimental process in many neurodegenerative disorders.Notably,our experimental results demonstrate high conformational sensitivity by differentiating subtle secondary-structural variations in a nativeβ-sheet protein monolayer from those of crossβ-sheets,which are characteristic of pathological aggregates.Our nanoplasmonic biosensor is a highly promising and versatile tool for in vitro structural analysis of thin protein layers.

Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus （wssv） and its use as a biosensor

White spot syndrome virus （WSSV） is a major cause of high mortality in cultured shrimp all over the world. VP26 is one of the structural proteins of WSSV that is assumed to assist in recognizing its host and assists the viral nucleocapsid to move toward the nucleus of the host cell. The objective of this work was to produce a polyclonal antibody against VP26 and use it as a biosensor. The recombinant VP26 protein （rVP26） was produced in E. coli （BL21）, purified and used for immunizing rabbits to obtain a polyclonal antibody. Western blot analysis confirmed that the antiserum had a specific immunoreac- tivity to the VP26 of WSSV. This VP26 antiserum was immobilized onto a gold electrode for use as the sensing surface to detect WSSV under a flow injection system. The impedance change in the presence of VP26 was monitored in real time. The sensitivity linear range of 160 160000 of the biosensor was in the copies of WSSV, indicating that it is good and sensitive for analysis of WSSV. The specificity of the biosensor was supported by the observation that no impedance change was detected even at high concentrations when using Yellow Head Virus （YHV）. This biosensor may be applied to monitor the amount of WSSV in water during shrimp cultivation.

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.