Comparison of two optical techniques for label-free detection of biomolecular microarrays on solids

I compare two techniques for label-free detection of biomolecular reactions on solid supports in microarray format. I show that the oblique-incidence reflectivity difference (OI-RD), when operated near the Brewster angle, is as sensitivity as the surface plasmon resonance technique. The OI-RD technique is more versatile and promises higher throughputs.     

用斜入射光反射差法无标记实时监测不同浓度兔IgG和山羊抗兔IgG反应的动力学过程

用斜入射光反射差法无标记实时监测1.25 mg/mL, 2.50 mg/mL和5.00 mg/mL三种不同浓度兔IgG和浓度为0.02 mg/mL山羊抗兔IgG反应的动态过程, 同时监测和获得三种不同浓度IgG反应的动力学曲线以及相应的反应时间. 实验结果表明, 斜入射光反射差法不仅能无标记地判别生物分子之间是否发生反应, 而且能无标记实时监测生物分子反应的动力学过程, 在生物分子相互作用的研究方面具有潜在和广泛的应用前景.     

Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5′-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3′, and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5′-TTC GAC CTC GGT TAG AAG ACT CAT-3′ and two-base mismatched DNA, 5′-TTC GAC AGC GGT TAT AAG ACT CAT-3′.     

Label-free high-throughput and real-time detections of protein interactions by oblique-incidence reflectivity difference method

Selected Mouse IgG of 1 mg/mL as target was fabricated on microarray for 500 sample dots.Label-free and real-time reaction dynamic processes were detected between the microarrays with Goat Anti-mouse IgG of 0.02 mg/mL using the obliqueincidence reflectivity difference(OIRD)method.We obtained the reaction results and the reaction dynamic curves of 500 protein dots.In addition,we also used label-free detection of protein microarrays of 10080 sample dots,including BSA and different concentrations of Mouse IgG and Rabbit IgG,by OIRD.The obtained reaction results between the protein microarray with 1 mg/mL Goat Anti-mouse IgG and 1 mg/mL Goat Anti-rabbit IgG are reported herein.Experimental results show that OIRD can be not only label-free high-throughput detection method for biological microarrays but also label-free real-time detection in the interaction processes of biomolecules.     

Detection of magnetic-labeled antibody specific recognition events by combined atomic force and magnetic force microscopy

Atomic force (AFM) and magnetic force microscopy (MFM) were developed to detect biomolecular specific interaction. Goat anti-mouse immunoglobulin (anti-IgG) was covalently attached onto gold substrate modified by a self-assembly monolayer of thioctic acid via 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) activation. Magnetic-labeled IgG then specifically adsorbed onto anti-IgG surface. The morphological variation was identified by AFM. MFM was proved to be a fine assistant tool to distinguish the immunorecognized nanocomposites from the impurities by detection of the magnetic signal from magnetic-labeled IgG. It would enhance the understanding of biomolecular recognition process.     

An integrated Mach–Zehnder interferometric biosensor with a silicon oxynitride waveguide by plasma-enhanced chemical vapor deposition

We report the design and fabrication of an integrated Mach–Zehnder interferometric (MZI) biochip based on silicon oxynitride layers deposited with a plasma-enhanced chemical vapor deposition (PECVD) process. A rib waveguide for an integrated MZI sensor has been designed to have a high surface sensitivity and a single-mode behavior by using an effective index method. The integrated MZI chip operating at 637 nm is fabricated via conventional photolithography and reactive ion etching. As a biosensor application, the real-time and label-free detection of the covalent immobilization and hybridization of DNA strands is performed and verified with this device.     

Overview of CMOS image sensor use in molecular diagnostics

CMOS sensors comprise an important tool in bioscientific applications. This review focuses on CMOS sensor-based molecular diagnostics of DNA, protein, and metabolic molecules. Herein, gene sequencing, DNA–DNA hybridization, single nucleotide polymorphisms (SNP), protein interactions, peptide interactions, antigen–antibody (Ag–Ab) interactions, as well as glucose and cholesterol monitoring using CMOS sensors are discussed along with existing experimental outcomes. CMOS sensor based electrochemical, optical, impedance, dual, continuous, and label-free analysis and their related integration techniques are explained. Moreover, we describe the utilization of a CMOS chip in microarray fabrication, assay platform development, and transducer incorporation for molecular diagnostics. Furthermore, CMOS sensor-based point-of-care (POC) applications, other biological analyses, and the role of nanoparticles in biomolecular sensing are discussed. Future directions include information about the novel integration of CMOS sensor-based molecular diagnostic devices with a central focus towards enhancement of POC approaches. This review is helpful in creating highly sensitive, cheaper, and user-friendly biomedical devices with modern dimensions.     

Surface energy and hybridization studies of amorphous carbon surfaces

Surface properties of a large number of amorphous carbon (a-C) films have been investigated using contact angle measurements and X-ray photoelectron spectroscopy (XPS). Dense a-C surfaces with variable sp³/(sp² + sp³) average hybridization were grown using sputtering or pulsed laser deposition (PLD) and were further chemically modified by thermal annealing, ion bombardment or covalent grafting of organic monolayers. The average carbon hybridization, impurity level and mass density, were deduced from XPS and photoelectron energy loss spectroscopy (PEELS). The depth sensitivity of the dispersive (Lifshitz–van der Waals) interaction, estimated at 1–2 nm from the dependence of γ^LW on the grafted perflorodecene molecule coverage, is much better than XPS which probes a 3–5 nm depth. The observation of a non-monotonic behavior in the correlation between surface hybridization and electron donor component of surface energy reveals that the average carbon hybridization alone does not describe the entire surface energy physics. The role of π bond clustering in the polar interactions is thus considered and some implications on surface reactivity and mutual interactions with molecular or biomolecular species are discussed.     

Gastric cancer screening by combined assay for serum anti-Helicobacter pylori IgG antibody and serum pepsinogen levels - "ABC method"

The current status of screening for gastric cancer-risk (gastritis A, B, C, D) method using combined assay for serum anti-Helicobacter pylori (Hp) IgG antibody and serum pepsinogen (PG) levels, "ABC method", was reviewed and the latest results of our ongoing trial are reported. It was performed using the following strategy: Subjects were classified into 1 of 4 risk groups based on the results of the two serologic tests, anti-Hp IgG antibody titers and the PG I and II levels: Group A [Hp(-)PG(-)], infection-free subjects; Group B [Hp(+)PG(-)], chronic atrophic gastritis (CAG) free or mild; Group C [Hp(+)PG(+)], CAG; Group D [Hp(-)PG(+)]), severe CAG with extensive intestinal metaplasia. Continuous endoscopic follow-up examinations are required to detect early stages of gastric cancer. Asymptomatic Group A, which accounts for 50-80% of all the subjects may be excluded from the secondary endoscopic examination, from the viewpoint of efficiency. Hp-infected subjects should be administered eradication treatment aimed at the prevention of gastric cancer.     

基于图像亮度特性统计的水下距离选通成像自动增益控制方法

对水下激光距离选通成像质量与系统增益的关系进行了分析,提出了一种基于图像亮度特性统计的普适性自动增益控制方案。该方案通过设定不同灰度统计阈值,甄别目标图像的亮度特性,并以此作为控制系统增益的主要依据。在对水下大动态范围照度目标或慢速运动目标等进行成像的特殊场合,该方案也具备较强的适用性,能实时反馈控制系统增益,使成像质量优化。     

免疫球蛋白G的免疫共振散射光谱分析

在pH 7.0 Tris-HCl缓冲溶液中及聚乙二醇-20000存在下,羊抗兔IgG与兔IgG的免疫复合物可聚集形成疏水的免疫复合物微粒,在330, 400, 520 nm处有三个共振散射峰,在470 nm有一个同步散射峰。IgG浓度在1.33～133.3 μg·mL-1范围内与470 nm处的散射强度呈线性。借此用于定量分析血清IgG, 结果满意。方法检出限为0.99 μg·mL-1。     

基于表面等离子体共振的新型超宽带微结构光纤传感器研究

基于表面等离子体共振的微结构光纤传感器具有高灵敏、免标记和实时监控等优点.如今,由于此类传感器广泛应用于食品安全控制、环境检测、生物分子分析物检测等诸多领域而受到大量研究.然而,目前所报道的绝大多数此类传感器只能应用于可见光或近中红外传感.因此,对可应用于中红外传感的表面等离子体共振微结构光纤传感器的研究是一项极具挑战性的工作.基于此,本文设计了一种可以工作在近红外和中红外区域的新型高灵敏表面等离子体共振微结构光纤传感器.传感器采用双芯单样品通道结构,该结构不仅可以消除相邻样品通道间的相互干扰和提高传感器的信噪比,还可以在超宽带波长范围内实现高灵敏检测.采用全矢量有限元法对其传感特性进行了系统的研究,研究结果表明:当待测样品折射率分布在1.423—1.513范围内时,传感器不仅可以在1.548—2.796μm的超宽带波长范围内进行工作,而且其平均灵敏度高达13964 nm/RIU.此外,传感器的最高波长灵敏度和折射率分辨率分别为17900 nm/RIU,5.59×10^–7 RIU.     

Rapid and label-free detection of H5N1 virus using carbon nanotube network field effect transistor

DNA hybridization-based detection techniques are widely used in genetics, medicine, and drug discovery. However, the current techniques are usually based on labels and reagents that are time consuming and complex to implement. In this study, we report a label-free DNA sensor based on single-walled carbon nanotube field effect transistor (SWCNTFET) for selective DNA hybridization detection of H5N1 virus. A network of single-walled carbon nanotubes (SWCNTs) acts as the conductor channel. Probe DNA sequences were adsorbed onto SWCNTs. The developed DNA sensor can effectively detect full-complementary DNA with concentration as low as 1.25 pM. The sensitivity of the DNA sensor reached approximately 0.28 nM/nA. The effect of the parameters, including DNA probe concentration, its complementary concentration, mismatched sequence, and hybridization time, on the sensor response was also studied. The results showed the potential application of the DNA sensor for medical, environmental, and epidemic detection.     

Homogeneous Phosphodiesterase and Hybridization Assays Using Europium Cryptate: Oligonucleotide Conjugates

Upon conjugation to single-stranded oligonucleotides, a europium cryptate (Eu³⁺ tris-bipyridine) showed a marked increase in its fluorescence lifetime and was much less sensitive to fluorescence quenching by uric acid. This behavior was shown to be moderately dependent on the length and sequence of the oligonucleotide and all the single-stranded oligonucleotides studied displayed similar behavior. In contrast, a cryptate moiety attached to a double-stranded oligonucleotide did not display such an increase in its fluorescence lifetime and was quenched in presence of uric acid. Taking advantage of this unique behavior characterizing single-stranded K-ODN conjugates, a new concept of dosage based on the modulation of the cryptate fluorescence by a quencher was set up. This fluorescence quenching assay involving a single fluorescent label was applied to the monitoring of hybridization reactions and detection of a phosphodiesterase activity.     

Increased yield of immunogold labeling of epoxy sections by adding para-phenylendiamine in the tissue processing

The purpose of this study was to examine if the presence of para-phenylendiamine (PPD) in the tissue processing could increase the yield of immunogold labeling of the epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. PPD was added (1) at the beginning of the dehydration, (2) in the first step with propylene oxide, (3) in the beginning of the dehydration and in all steps with propylene oxide included the infiltration step where propylene oxide and epoxy resin are mixed, or (4) PPD was totally avoided. The tissue was embedded with two different combinations of accelerator. Immunogold labeling with anti-IgG was performed on both non-heated and heated ultrathin sections. The immunogold labeling on the heated sections which were based on processing with PPD in all steps (3) was about 55–65% higher than the corresponding labeling for epoxy sections processed in total absence of PPD (4). The immunolabeling was not significantly increased when the tissue was processed with PPD only in the start of the dehydration (1) or in the first step with propylene oxide (2). We believe that tissue processing with sufficient PPD contributes to reduce the co-polymerization between the antigens and the epoxy polymer in the same way as excess of accelerator does (Brorson and Skjørten, 1996a). The practical significance of this study provides better opportunities for increasing the immunogold labeling of epoxy sections by adding PPD in the tissue processing, and our result may inspire other researchers to develop even more efficient methods for controlling the copolymerization between antigens and epoxy resin.     

压电基因传感器研究进展

压电基因传感器是一种新型的生物传感器，它将压电传感器的灵敏性和DNA杂交反应结合在一起。与传统的基因检测技术相比，压电基因传感器具有结构简单、不需要标记、检测时间短等特点。本对压电基因传感器的研究现状作了综述，包括压电基因传感器的原理、测量技术、固定方法、以及压电基因传感器在各个领域的应用。     

Optical heterodyne surface-plasmon resonance biosensor

A novel optical heterodyne surface-plasmon resonance (SPR) biosensor with a Zeeman laser is proposed. Two surface plasma waves are excited by two correlated p-polarized waves in a SPR device of the Kretschmann configuration. Two reflected p waves are optically heterodyned such that the magnitude of the heterodyned signal is proportional to the multiplication of two attenuated reflected p waves. Then the detection sensitivity and the dynamic range based on this amplitude-sensitive method are enhanced. In the experiment, the kinetics between mouse immunoglobulin G (IgG) and rabbit antimouse IgG is obtained from sensograms of various concentrations of antimouse IgG. A detection sensitivity of 0.2 nM was achieved. In addition, a concentration of 5 ng/ml of protein G interacting with mouse IgG was measured successfully.     

用光波导光模光谱技术研究DNA-DNA结合蛋白相互作用

探讨了应用光波导光模光谱(Optical waveguide lightmode spectroscopy,OWLS)技术研究DNA-DNA结合蛋白相互作用的可行性和灵敏性。以固定在传感器芯片表面的DNA探针为捕捉分子,溶液中同时含有探针结合序列和NF—κB结合位点序列的寡核苷酸与NF-κB亚单位p50同源二聚体形成的DNA-蛋白质复合物为检测分子,用光波导光模光谱检测技术建立非标记DNA-DNA结合蛋白相互作用检测研究体系。利用这一体系对不同样品中NF-κB p50浓度和具不同NF-κB结合位点序列的寡核苷酸与NF-κB p50亲合和力进行检测。样品中低至0．33 nmol/1的NF-κB p50被光波导光模光谱检测出,不同的NF-κB结合序列与NF-κB p50亲合力有显著的差异。研究发现,光波导光模光谱技术可以用于DNA-DNA结合蛋白相互作用研究,所建立的非标记检测研究体系可以进行样品中结合蛋白含量高灵敏检测和核酸序列与结合蛋白的亲合力的检测研究。     

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.