A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.     

A single- and a dual-fractal analysis of antigen-antibody binding kinetics for different biosensor applications.

The diffusion-limited binding kinetics of antigen (or antibody) in solution to antibody (or antigen) immobilized on a biosensor surface is analyzed within a fractal framework. The data is adequately described by a single- or a dual-fractal analysis. Initially, the data was modelled by a single-fractal analysis. If an inadequate fit was obtained then a dual-fractal analysis was utilized. The regression analysis provided by Sigmaplot, 1993 (Scientific Graphing Software: User's Manual. Jandel Scientific, San Rafael, CA) was utilized to determine if a single-fractal analysis is sufficient, or a dual-fractal analysis is required. In general, it is of interest to note that the binding rate coefficient and the fractal dimension exhibit changes in the same direction (except for a single example) for the antigen-antibody systems analyzed. Binding rate coefficient expressions as a function of the fractal dimension developed for the antigen-antibody binding systems indicate a high sensitivity of the binding rate coefficient on the fractal dimension when both a single -as well as a dual-fractal analysis is used. For example, for a single-fractal analysis and for the binding of human endothelin-1 (ET-1) antibody in solution to ET-1(15-21) x BSA (bovine serum albumin) immobilised on a surface plasmon resonance surface, the order of dependence of the binding rate coefficient, k on the fractal dimension, Df is 7.0945. Similarly, for a dual-fractal analysis and for the binding of parasite L. donovani diluted pooled sera in solution to fluorescein isothiocyanate-labeled anti-human immunoglobulin IgG immobilized on an optical fibre, the order of dependence of k1 and k2 on Df1 and Df2 were 6.8018 and -4.393, respectively. Binding rate coefficient expressions are also developed as a function of the analyte (antigen or antibody) concentration in solution. The binding rate coefficient expressions developed as a function of the fractal dimension(s) are of particular value since they provide a means to better control biosensor performance by linking it to the heterogeneity on the surface, and emphasize in a quantitative sense the importance of the nature of the surface in biosensor performance.     

On antigen-antibody binding distribution

The probability distributions for the number of bound antigens and antibodies during immune response are obtained in this paper. Biological significance of this work and directions for further application are discussed along with some illustrative numerical results.     

Rapid self-assembly of DNA on a microfluidic chip

Background  

DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation.     

Electrochemical modulation of antigen-antibody binding

The label-free amperometric detection of a rabbit IgG antigen by an anti-rabbit IgG antibody is achieved by observing the electrochemistry at a glassy carbon electrode modified with antibody entrapped in an electrodeposited polypyrrole membrane. In a flow injection apparatus the electrode is pulsed between -0.2 and +0.4 V versus Ag/AgCl. The pulsing of the electrode switches the polypyrrole membrane between the oxidised and reduced states. When antigen is injected into the flow stream a change in current is observed at the electrode despite the antigen or antibody being redox inactive at the potentials employed. It is proposed that this current is due to a change in the flux of ions into and out of the polypyrrole matrix during a pulse when the poly-anionic antigen is present. The immunoreaction was reversible because the 200 ms pulse at each potential was too short to allow secondary bonding forces (hydrogen bonding and hydrophobic forces) which are responsible for the strength of the antibody-antigen complex to be established. The consequence of the reversibility of the antigen-antibody binding is a low apparent affinity constant but an easily regenerated recognition interface.     

Urinary proteome profiling using microfluidic technology on a chip

Clinical diagnostics and biomarker discovery are the major focuses of current clinical proteomics. In the present study, we applied microfluidic technology on a chip for proteome profiling of human urine from 31 normal healthy individuals (15 males and 16 females), 6 patients with diabetic nephropathy (DN), and 4 patients with IgA nephropathy (IgAN). Using only 4 microL of untreated urine, automated separation of proteins/peptides was achieved, and 1-7 (3.8 +/- 0.3) spectra/bands of urinary proteins/peptides were observed in the normal urine, whereas 8-16 (11.3 +/- 1.2) and 9-14 (10.8 +/- 1.2) spectra were observed in urine samples of DN and IgAN, respectively. Coefficient of variations of amplitudes of lower marker (1.2 kDa), system spectra (6-8 kDa), and upper marker (260.0 kDa) were 22.84, 24.92, and 32.65%, respectively. ANOVA with Tukey post-hoc multiple comparisons revealed 9 spectra of which amplitudes significantly differed between normal and DN urine (DN/normal amplitude ratios ranged from 2.9 to 3102.7). Moreover, the results also showed that 3 spectra (with molecular masses of 12-15, 27-28, and 34-35 kDa) were significantly different between DN and IgAN urine (DN/IgAN amplitude ratios ranged from 3.9 to 7.4). In addition to the spectral amplitudes, frequencies of some spectra could differentiate the normal from the diseased urine but could not distinguish between DN and IgAN. There was no significant difference, regarding the spectral amplitude or frequency, observed between males and females. These data indicate that the microfluidic chip technology is applicable for urinary proteome profiling with potential uses in clinical diagnostics and biomarker discovery.     

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.     

Rapid antibody selection by mRNA display on a microfluidic chip

In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10⁶- to 10⁸-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naïve and randomized single-chain Fv libraries of ~10¹² molecules. Furthermore, we confirmed that not only protein–protein (antigen–antibody) interactions, but also protein–DNA and protein–drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.     

基于微流控芯片的等温扩增技术北大核心CSCD

基于聚合酶链式反应(polymerase chain reaction,PCR)的核酸扩增技术是分子诊断领域的金标准,然而PCR往往包含多个反应温度,涉及长时间的循环升降温过程,且需要在复杂热循环仪中完成,这些都限制了其在现场即时检测(point-of-care testing,POCT)中的应用。与传统PCR相比,等温扩增依靠恒定反应温度,反应时间短,检测装置简单,能够提供更加方便、快捷的核酸检测。基于微流控技术的等温扩增检测,通过兼顾微流控与等温扩增两者的优势,能够为POCT分子诊断提供更具竞争力的平台。例如,在新型冠状病毒肺炎(COVID-19)疫情防控中,多种形式的POCT等温扩增检测展示了其独特优势。文中首先归纳总结了典型的等温扩增技术及其检测方法,然后对不同类型的等温扩增微流控系统进行了分类总结与分析(如功能定位、结构组成、流体控制、系统特点等),最后总结了等温扩增微流控系统在新冠病毒(SARS-CoV-2)等不同病原体检测领域中的应用,并对等温扩增与CRISPR基因编辑等其他新型技术的相互结合进行了介绍与展望。     

Determination of the binding parameters for a pre-existing antigen-antibody mixture

A new method, which allows to evaluate parameters of interaction between antibodies (or receptors) and an antigen (or ligand) is suggested. The method is based on the use of so-called coordinates of dilution suggested by the author earlier. Representation of the data of the titration curves for the mixtures of antibodies (or receptors) and antigen (or ligand) in these coordinates allows one to determine the affinity of interaction and the concentration of antigen (or ligand), which can reversibly block antibodies (or receptors). Simple formulas, which allow to estimate which part of paratopes or bivalent antibodies is free and which part is blocked by the antigen, depending on dilution of the considered system, are also suggested. Such a method could be useful for characterization of infection and autoimmune processes when the antigen and antibodies circulate together in the bloodstream.     

Numerical design of a microfluidic chip for probing mechanical properties of cells

Microfluidic chips have been widely used to probe the mechanical properties of cells, which are recognized as a promising label-free biomarker for some diseases. In our previous work (Ye et al., 2018), we have studied the relationships between the transit time and the mechanical properties of a cell flowing through a microchannel with a single constriction, which potentially forms a basis for a microfluidic chip to measure cell’s mechanical properties. Here, we investigate this microfluidic chip design and examine its potential in performances. We first develop the simultaneous dependence of the transit time on both the shear and bending moduli of a cell, and then examine the chip sensitivity with respect to the cell mechanical properties while serializing a single constriction along the flow direction. After that, we study the effect of the flow velocity on the transit time, and also test the chip’s ability to identify heterogeneous cells with different mechanical properties. The results show that the microfluidic chip designed is capable of identifying heterogeneous cells, even when only one unhealthy cell is included. The serialization of chip can greatly increase the chip sensitivity with respect to the mechanical properties of cells. The flow with a higher velocity helps in not only promoting the chip throughput, but also in providing more accurate transit time measurements, because the cell prefers a symmetric deformation under a high velocity.     

Microcomputer simulation of antigen-antibody binding at a fixed antigen/antibody ratio

Computer simulation techniques were used to define the theoretical nature of the reaction of antigen with antibody at a constant degree of antigen excess. The simulation studies show that binding curves obtained under these conditions have unique features which are determined by antibody affinity, affinity heterogeneity and the concentrations of antigen and antibody. When analyzed by non-linear least squares regression these curves can be used to determine the affinity of antibody in homogeneous and heterogeneous systems.     

聚合物微流控芯片消毒灭菌技术研究进展

聚合物微流控芯片成本低、易加工,目前在医药、生物检测和化学合成等领域得到了普遍应用。以热塑性聚合物聚甲基丙烯酸甲酯(polymethylmethacrylate,PMMA)和热固型聚合物聚二甲基硅氧烷(polydimethy lsiloxane,PDMS)为基材的高分子聚合物材料因具有较好的生物相容性和光学透明性,已逐渐成为聚合物微流控芯片加工的主导材料,被广泛应用于生物医药类微流控芯片的制备。鉴于该类芯片应用场景的特殊性,需在使用前进行消毒灭菌处理以避免微生物干扰。目前,针对PMMA和PDMS的消毒灭菌方法包括高压蒸汽灭菌、紫外线灭菌、电子束、60Co γ射线辐射灭菌、超临界二氧化碳灭菌、乙醇消毒、环氧乙烷灭菌、过氧化氢低温等离子体灭菌、绿原酸消毒、清洗剂消毒。本文从基本原理、消毒灭菌方法、应用场景等方面,回顾和总结了相关技术在PMMA和PDMS基体微流控芯片中的实现方法,并在芯片材质、适用范围等方面分析了所适用的消毒灭菌方法,为以聚合物为基材的生物医药类微流控芯片的消毒灭菌提供有益参考。     

Study of the kinetics of antigen-antibody reaction by light scattering

Laser light scattering in conjunction with measurement of the spectral width and integral intensity of light scattering was applied to studying the process of complex formation of antigens and antibodies. The system of polysaccharide group A streptococcus and antibody against it was examined under varying polysaccharide concentrations. The measurements were performed every 10 s for 70 min after combining polysaccharide and antibody solutions. Within the entire time interval, the size of the complexes were less than the wave length of exciting laser light (0.633 m). This made it possible to determine their average magnitude in terms of the Rayleigh model.     

Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and R9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.     

微流控芯片在中枢神经系统疾病中的应用与展望

近年来,随着微流体技术和生物微电子机械系统技术的不断发展,人类中枢神经系统(CNS)的微流体平台及相关疾病的体外模型逐渐得到了广泛的研究。微流体平台可以更好地模拟体内环境,同时能够控制结构、微环境和外来刺激。文中总结了微流控芯片在CNS的基本技术和CNS疾病中的应用。此外,文中对微流控芯片在CNS中的研究进行了展望,强调了通过跨学科的共同努力能够实现更高程度的仿生学挑战。