Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy

The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.Dedicated to the memory of Wilhelm Fresenius.     

Label-free and direct protein detection on 3D plasmonic nanovoid structures using surface-enhanced Raman scattering

In this paper we use surface-enhanced Raman spectroscopy (SERS) on 3D metallic structures for label-free detection and characterization of proteins of interest at low concentrations. The substrates are prepared via nanopatterning with latex nano/microparticles and Cr and Ag sputtering, yielding stable, tunable, and mechanically flexible plasmonic structures. The nanovoids generate a SERS signal of the proteins of interest that is background free and independent of the protein charge. Concentrations as low as 0.05 μg mL⁻¹ could be detected for 4 different proteins. The proteins also exhibit significantly different SERS spectra on these substrates, which is an important feature for future label-free direct detection schemes.     

Total internal reflectance fluorescence (TIRF) biosensor for environmental monitoring of testosterone with commercially available immunochemistry: antibody characterization, assay development and real sample measurements

Nowadays, little technology exists that can monitor various water sources quickly and at a reasonable cost. The ultra-sensitive, fully automated and robust biosensor River Analyser (RIANA) is capable of detecting multiple organic targets rapidly and simultaneously at a heterogeneous assay format (solid phase: bulk optical glass transducers). Commercialization of such a biosensor requires the availability of commercial high-affinity recognition elements (e.g. antibodies) and suitable commercial haptens (modified target molecules) for surface chemistry. Therfore, testosterone was chosen as model analyte, which is also a task of common analytical methods like gas chromatography-mass spectrometry (GC-MS), because they have to struggle with detecting sub-nanogram per liter levels in environmental samples. The reflectometric interference spectroscopy (RIfS) was used to characterize the commercially available immunochemistry resulting in a high-affinity constant of 2.6 ± 0.3 × 10⁹ mol⁻¹ for the unlabeled antibody. After the labeling procedure, necessary for the TIRF-based biosensor, a mean affinity constant of 1.2 × 10⁹ mol⁻¹ was calculated out of RIfS (1.4 ± 0.4 × 10⁹ mol⁻¹) and TIRF (1.0 ± 0.3 × 10⁹ mol⁻¹) measurements.Thereafter, the TIRF-based biosensor setup was used to determine the steroidal hormone testosterone at real world samples without sample pre-treatment or sample pre-concentration. Results are shown for rapid and ultra-sensitive analyses of testosterone in aqueous samples with at a remarkable limit of detection (LOD) of 0.2 ng L⁻¹. All real world samples, even those containing testosterone in the sub-nanogram per liter range (e.g. 0.9 ng L⁻¹), could be determined with recovery rates between 70 and 120%. Therefore, the sensor system is perfectly suited to serve as a low-cost system for surveillance and early warning in environmental analysis in addition to the common analytical methods. For the first time, commercially available immunochemistry was fully characterized using a label-free detection method (RIfS) and successfully incorporated into a TIRF-based biosensor setup (RIANA) for reliable sub-nanogram per liter detection of testosterone in aqueous environmental samples.     

Chemiluminescence detection of label-free C-reactive protein based on catalytic activity of gold nanoparticles

A novel, quantitative analytical method for measuring C-reactive protein (CRP) levels in human serum has been developed based on the catalytic activity of gold nanoparticles (GNPs) and luminol-H₂O₂ chemiluminescence (CL). The CL intensity in the presence of CRP and its ligand, O-phosphorylethanolamine (PEA), was greatly enhanced due to the aggregation of GNPs after the addition of 0.5 M NaCl. Any pretreatment steps, such as covalent functionalization of GNPs, addition of antibodies, or labeling of CRP, were not needed for CL detection. The CL enhancement was linearly proportional to CRP concentration in the range of 1.88 fM to 1.925 pM. The detection limit of CRP in serum samples was estimated to be as low as 1.88 fM. The detection sensitivity was increased more than 164 times of magnitude over that of the conventional, enzyme-linked immunosorbent assay (ELISA) method. This proposed GNP-based CL detection method offers the advantages of simplicity, rapidity, and sensitivity.     

基于Pd/COF-LZU1非标记型C-反应蛋白免疫传感器的研制

采用溶剂热法, 通过有机单体合成了一种亚胺键连接的共价有机框架材料(COF-LZU1); 在常温常压条件下, 通过后合成的方法将贵金属钯(Ⅱ)引入到COF材料中, 合成了复合材料Pd/COF-LZU1, 该材料具有优良的催化性能. 利用Pd/COF-LZU1多孔复合材料将C-反应蛋白(CRP)抗体(anti-CRP)固定在玻碳电极表面, 构建了一种非标记型CRP免疫传感器. 当抗体与抗原发生免疫反应时, 形成的免疫复合物会阻碍电化学探针[Fe(CN)₆]^4-/3-的电子传递, 降低其响应电流, 从而实现CRP的快速检测. 采用交流阻抗和差示脉冲伏安法(DPV)考察了免疫传感器的电化学特性, 同时考察了测试底液的pH值、 抗原培育时间和抗体固定浓度等实验条件对传感器性能的影响. 在最优的实验条件下, 采用DPV法对CRP进行检测的线性范围为5~180 ng/mL, 检出限为1.66 ng/mL, 线性相关系数为0.992.     

Selective detection and estimation of C-reactive protein in serum using surface-functionalized gold nano-particles

A new method for the detection of C-reactive protein (CRP) in serum using functionalized gold nano-particles (GNP) is reported. The affinity towards CRP is imparted to GNP by tethering O-phosphorylethanolamine (PEA) onto their surface. GNP and modified GNP were characterized using TEM, particle size analysis, zeta potential measurements, absorption spectroscopy and FT-IR techniques. The event of binding of CRP onto the PEA-GNP is followed by visibly observable colour change. We observed a red shift as well as a decrease in absorption in the plasmon peak of the modified GNP with the concentration of CRP. When the concentration of CRP exceeded 450 ng mL⁻¹, particles were aggregated and the solution became turbid. The method exhibited a linear range for CRP from 50 to 450 ng mL⁻¹ with a detection limit of 50 ng mL⁻¹. The colour change and the variation in absorption of the GNP were highly specific to CRP even in the presence of albumin. We estimated CRP in blood serum collected from patients and the results obtained compared well with the estimation using the technique of nephelometry based on the antibody-antigen interaction.     

Characterisation of morphology of self-assembled PEG monolayers: a comparison of mixed and pure coatings optimised for biosensor applications

For detection of low concentrations of analytes in complex biological matrices using optical biosensors, a high surface loading with capture molecules and a low nonspecific binding of nonrelevant matrix molecules are essential. To tailor biosensor surfaces in such a manner, poly(ethylene glycols) (PEG) in varying lengths were immobilised covalently onto glass-type surfaces in different mixing ratios and concentrations, and were subsequently modified with three different kinds of receptors. The nonspecific binding of a model protein (ovalbumin, OVA) and the maximum loading of the respective analytes to these prepared surfaces were monitored using label-free and time-resolved reflectometric interference spectroscopy (RIfS). The three different analytes used varied in size: 150 kDa for the anti-atrazine antibody, 60 kDa for streptavidin and 5 kDa for the 15-bp oligonucleotide. We investigated if the mixing of PEG in different lengths could increase the surface loadings of analyte mimicking a three-dimensional matrix as was found using dextrans as sensor coatings. In addition, the effect on the surface loading was investigated with regard to the size of the analyte molecule using such mixed PEGs on the sensor surface. For further characterisation of the surface coatings, polarisation modulation infrared reflection absorption spectroscopy, atomic force microscopy, and ellipsometry were applied. All authors contributed equally to this work.     

Versatile biosensor surface based on peptide nucleic acid with label free and total internal reflection fluorescence detection for quantification of endocrine disruptors

In recent years, the simultaneous detection of hundreds of substances has become increasingly important in medical diagnostics and environmental monitoring. This calls for methods that allow fast and simultaneous measurement of many analytes and require only a minimum of attendance. Biosensor-applications including easy implementation and long-term stability of the sensor element are more and more designed to meet these demands.In this paper, a surface based on covalently immobilised peptide nucleic acid (PNA) for the detection of different endocrine disruptors is used. The surface was characterised with a label free detection system, the reflectometric interference spectroscopy (RIfS). A hybridisation capacity with DNA oligonucleotides of 1.3 ng/mm² (180 fmol/mm²) on PNA-surfaces was achieved. The PNA transducer is stable for half a year and for more than 300 regeneration steps.With this modified surface various environmentally relevant endocrine disruptors could be detected by an immunoassay. The detection is done on an optical waveguide system, based on total internal reflection fluorescence (TIRF) by using an auxiliary-system consisting of a conjugate which is formed of DNA-oligomers and analyte-derivatives. The successive detection of different analytes on the same spot by using this auxiliary-system is demonstrated as one application of multianalyte detection. Quantification of different endocrine disruptors on the same PNA-surface is demonstrated by three calibration curves.     

Nanomagnet Bioconjugates with anti-CRP Polyclonal Antibodies as Nanobioprobes for Enhanced Impedimetric Detection of CRP

Selective purification of biological materials for their detection in complex sample matrix is a general challenge for many researchers working in the field of diagnostics. Magnetic nanoparticles functionalized with biological molecules that impart biomolecular selectivity are therefore of major interest as capture probes thus allowing for magnetic separations. Nanoparticles can also be used for the enhanced detection of biomarkers. In this work, an ultrasensitive sandwich-based impedimetric immunosensor was fabricated for the detection of C-reactive protein antigen (CRPAg). Stable and oriented immobilization of anti-CRP monoclonal antibody was achieved onto electrografted phenylethylamine derivatized with succinic anhydride and phenylboronic acid via carbodiimide chemistry. The detection of CRPAg was achieved using Electrochemical Impedance Spectroscopy (EIS). The enhancement of the impedance charge-transfer resistance (R_CT) signal was achieved using the sandwich approach. The anti-CRP polyclonal antibody was immobilized in an oriented manner onto magnetic nanoparticles functionalized with phenylboronic acid. The increase in the change in charge-transfer resistance (ΔR_CT) values was linearly proportional to the concentration of CRPAg in the range 10 to 200 ng mL⁻¹ covering the clinical range for CRPAg detection and with a detection limit of 0.34 ng mL⁻¹.     

Label-free detection of the aptamer binding on protein patterns using Kelvin probe force microscopy (KPFM)

Anti-lysozyme aptamers are found to preferentially bind to the edge of a tightly packed lysozyme pattern. Such edge-binding is due to the better accessibility and flexibility of the edge lysozyme molecules. Kelvin probe force microscopy (KPFM) was used to study the aptamer–lysozyme binding. Our results show that KPFM is capable of detecting the aptamer–protein binding down to the 30 nm scale. The surface potential of the aptamer–lysozyme complex is approximately 12 mV lower than that of the lysozyme. The surface potential images of the aptamer-bound lysozyme patterns have the characteristic shoulder steps around the pattern edge, which is much wider than that of a clean lysozyme pattern. These results demonstrate the potentials of KPFM as a label-free method for the detection of protein–DNA interactions. Figure Aptamers preferentially bind on the edge of a protein pattern as revealed by Kelvin force microscopy.

高效液相色谱-二极管阵列检测法及高效液相色谱-电喷雾串联质谱法测定植物源性蛋白中残留的三聚氰胺

建立了高效液相色谱-二极管阵列检测器(HPLC-DAD)及HPLC-电喷雾串联质谱(ESI-MS/MS)测定植物源性蛋白中残留的三聚氰胺的方法。利用HPLC-DAD进行样品中三聚氰胺的初筛,利用HPLC-MS/MS进行确证。采用三氯乙酸溶液沉淀样品中的蛋白,同时提取目标分析物,质谱检测时样品再经强阳离子固相萃取柱富集净化。HPLC-DAD的检测低限为10 mg/kg,HPLC-MS/MS的检测低限为0.5 mg/kg;HPLC-DA的添加回收率为76%～88%,HPLC-MS/MS的添加回收率为72%～82%(基质匹配曲线校正),两种方法的添加回收率的相对标准偏差(RSD)为3.4%～6.4%。