Electrical impedance assays of blood cells

In this review, the capability of electrical impedance spectroscopy analysis of blood cells, especially for red blood cells is presented, highlighting its large area of related biomedical relevance. The method is briefly introduced and basic theoretical aspects are discussed by considering both phenomenological (e.g. equivalent circuit) and microscopic approaches. The latter include a comparative analysis of the relevance of considering real shape (consistent with microscopic observations) versus spheroidal approximations (prolate and oblate spheroids) with the same surface and volume concentration. We show that while ellipsoidal approximation is fairly good for randomly oriented cells, it is quite poor whenever oriented cells are measured. The voluminous literature on the electrical analysis of blood cells is reviewed to stress the most promising biomedical applications of the method either per se or in combination with complementary e.g. (micro) fluidic approaches.     

纳米多孔硅阻抗生物传感器的研究

构建了1种基于多孔硅材料,无需标记的纳米生物传感器,用于对牛血清白蛋白分子进行检测。通过对多孔硅进行表面处理,形成氧化膜,将抗体固定到多孔硅氧化层表面。在磷酸盐缓冲液中,通过电化学检测系统检测加入抗原后,传感器的阻抗值的变化。磷酸缓冲液（PBS）/抗体-氧化层/硅,构成电解液/绝缘层/半导体（electro-lyte-insulator-semiconductor,EIS）结构。传感器的线性检测范围为0.01～0.27mg/mL,检测限为0.01mg/mL。     

Formulation, characterization, and in vitro evaluation of quantum dots loaded in poly(lactide)-vitamin E TPGS nanoparticles for cellular and molecular imaging

We developed a novel system of poly(lactide acid)-d-alpha-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) nanoparticles (NPs) for quantum dots (QDs) formulation to improve imaging effects and reduce side effects as well as to promote a sustainable imaging. The QDs-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method, which were then characterized by laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and transmission electron microscope (TEM) for surface morphology. Surface chemistry of the QDs-loaded PLA-TPGS NPs was analyzed by X-ray photoelectron microscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR). Encapsulation efficiency of the QDs in the polymeric nanoparticles was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The photostability of the QDs formulated in the PLA-TPGS nanoparticles was investigated as changes in the florescence intensity versus the irradiation time. Confocal laser scanning microscopy (CLSM) was used to image the cellular uptake of the QDs-loaded NPs by MCF-7 cells. Methylthiazolyldiphenyl-tetrazolium (MTT) assay was employed to assess the viability of MCF-7 cells incubated with the QDs formulated by the PLA-TPGS NPs versus the mercaptoacetic acid (MAA)-coated QDs. It was found that the QDs formulated in the PLA-TPGS NPs can result in higher fluorescence intensity and higher photostability than the bare QDs as well as lower cytotoxicity than the MAA-coated QDs.     

光学显微镜技术在活细胞单分子检测中的应用

单分子检测是一门以高度的时间以及空间分辨率研究生物单分子的技术。近来,科学技术的探索发展使我们可以观察、检测甚至操纵单个分子并且研究它们的构象变化和动力学行为。这一发展使得以前被传统系综研究体系平均化所隐藏的新信息被揭示出来。单分子检测技术的发展已经揭开了生命科学研究的新篇章。在本文中,我们将介绍有关活细胞中单分子检测技术的发展以及活细胞内单分子检测的现状。     

Real time detection and quantification of inclusion bodies expressed in Escherichia coli by impedance measurements

Escherichia coli expressing human growth hormone as inclusion bodies was cultured in a fermenter. Real time, non-invasive detection and quantification of inclusion body protein expressed in E. coli was performed by impedance measurements at 50 MHz and 180 MHz. At 50 MHz rotation of dipoles of the protein and their proton fluctuation, i.e., -dispersion of protein aggregates formed inside the cell as a result of expressed protein, results in an additional decrease in impedance. At 180 MHz the impedance remained at a plateau. In a high cell density E. coli culture, after induction with IPTG, when the cell mass remained unchanged, an increase in the magnitude of -dispersion was observed at 50 MHz. This was due to the formation and subsequent increase in the concentration of r-human growth hormone which aggregate as inclusion bodies. The estimation of inclusion bodies by taking the ratio of impedance at 180 MHz and at 50 Mhz matched with the amount of protein estimated after extraction and purification (coefficient of correlation was 0.92). This is the first report of real time detection and monitoring of recombinant protein expressed as inclusion bodies by impedance measurements.     

Changes in electrical impedance of the vaginal medium during the menstrual cycle of female rhesus monkeys (Macaca mulatta)

Changes in electrical impedance of the vaginal medium during the menstrual cycle were recorded in female Rhesus monkeys using electrical probe and were correlated with estradiol-17 beta and progesterone plasma concentrations. A gradual decrease in impedance was observed during the follicular phase, the lowest values being observed between days 12-17 of the cycle. Impedance increased again during the first third of the luteal phase until day 21. The reversal of the impedance gradient's sign was nearly concomitant with the appearance of a detectable plasma progesterone concentration. These results support the use of vaginal impedance measurements as a help for the diagnosis of the periovulatory time in the female Rhesus monkey.     

脑电图机电极与头皮接触阻抗的检测

脑电图机中电极与头皮接触的好坏对脑电波形质量有很大影响，本文利用AT89C51单片机实现电极与头皮接触阻抗的检测。还通过发光二极管给予医务人员对电极接触好坏直观的指示。     

Direct and label‐free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor

A label‐free interferometric transducer showing a theoretical detection limit for homogeneous sensing of 5 × 10^–8 RIU, being equivalent to a protein mass coverage resolution of 2.8 fg mm^–2, is used to develop a high sensitive biosensor for protein detection. The extreme sensitivity of this transducer combined with a selective bioreceptor layer enables the direct evaluation of the human growth hormone (hGH) in undiluted urine matrix in the 10 pg mL^–1 range.

     

Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria

The realization of rapid, sensitive, and specific methods to detect foodborne pathogenic bacteria is central to implementing effective practice to ensure food safety and security. As a principle of transduction, the impedance technique has been applied in the field of microbiology as a means to detect and/or quantify foodborne pathogenic bacteria. The integration of impedance with biological recognition technology for detection of bacteria has led to the development of impedance biosensors that are finding wide-spread use in the recent years. This paper reviews the progress and applications of impedance microbiology for foodborne pathogenic bacteria detection, particularly the new aspects that have been added to this subject in the past few years, including the use of interdigitated microelectrodes, the development of chip-based impedance microbiology, and the use of equivalent circuits for analysis of the impedance systems. This paper also reviews the significant developments of impedance biosensors for bacteria detection in the past 5 years, focusing on microfabricated microelectrodes-based and microfluidic-based Faradaic electrochemical impedance biosensors, non-Faradaic impedance biosensors, and the integration of impedance biosensors with other techniques such as dielectrophoresis and electropermeabilization.     

细胞活性检测方法之比较

细胞活性的检测是体外细胞培养技术中常用的一个检测指标。常用的方法有直接染色法、克隆形成试验、比色法等。概述了一些常用的细胞活性检测方法的原理、特点,并对比了各自的优缺点。     

Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy

Three-dimensional (3D) cell culture has developed rapidly over the past 5–10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.     

Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.     

Circulating tumor cells in the early detection of human cancers

Cancer is a severe disease with high morbidity and mortality globally. Thus, early detection is emerging as an important topic in modern oncology. Although the strategies for early detection have developed rapidly in recent decades, they remain challenging due to the subtle symptoms in the initial stage of the primary tumor. Currently, tumor biomarkers, imaging, and specific screening tests are widely used in various cancer types; however, each method has limitations. The harms are even overweight against the benefits in some cases. Therefore, early detection approaches should be improved urgently. Liquid biopsy, for now, is a convenient and non-invasive way compared to the traditional tissue biopsy in screening and early diagnosis. Circulating tumor cells (CTCs) are vital in liquid biopsy and play a central role in tumor dissemination and metastases. They have promising potential as cancer biomarkers in early detection. This review updates the knowledge of the biology of CTC; it also highlights the CTC enrichment technologies and their applications in the early detection of several human cancers.     

Continuous flow system for biofilm formation using controlled concentrations of Pseudomonas putida from chicken carcass and coupled to electrochemical impedance detection

Abstract

Most studies dealing with monitoring the dynamics of biofilm formation use microbial suspensions at high concentrations. These conditions do not always represent food or water distribution systems. A continuous flow system capable of controlling the concentration of the microbial suspension stream from 10⁴ to 10⁶ CFU ml^?1 is reported. Pseudomonas putida biofilms formed using 100-fold, 1,000-fold or 10,000-fold diluted bacterial suspensions were monitored in-line by electrochemical impedance spectroscopy (EIS) and total plate counts. Randles equivalent circuit model and a modified Randles model with biofilm elements were used to fit the EIS data. In Randles equivalent circuit, the charge transfer resistance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the charge transfer resistance. In the biofilm model, the biofilm resistance and the double layer capacitance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the biofilm resistance.     

Impact of initial biofilm growth on the anode impedance of microbial fuel cells

Electrochemical impedance spectroscopy (EIS) was used to study the behavior of a microbial fuel cell (MFC) during initial biofilm growth in an acetate-fed, two-chamber MFC system with ferricyanide in the cathode. EIS experiments were performed both on the full cell (between cathode and anode) as well as on individual electrodes. The Nyquist plots of the EIS data were fitted with an equivalent electrical circuit to estimate the contributions of various intrinsic resistances to the overall internal MFC impedance. During initial development of the anode biofilm, the anode polarization resistance was found to decrease by over 70% at open circuit and by over 45% at 27 microA/cm(2), and a simultaneous increase in power density by about 120% was observed. The exchange current density for the bio-electrochemical reaction on the anode was estimated to be in the range of 40-60 nA/cm(2) for an immature biofilm after 5 days of closed circuit operation, which increased to around 182 nA/cm(2) after more than 3 weeks of operation and stable performance in an identical parallel system. The polarization resistance of the anode was 30-40 times higher than that of the ferricyanide cathode for the conditions tested, even with an established biofilm. For a two-chamber MFC system with a Nafion 117 membrane and an inter-electrode spacing of 15 cm, the membrane and electrolyte solution dominate the ohmic resistance and contribute to over 95% of the MFC internal impedance. Detailed EIS analyses provide new insights into the dominant kinetic resistance of the anode bio-electrochemical reaction and its influence on the overall power output of the MFC system, even in the high internal resistance system used in this study. These results suggest that new strategies to address this kinetic constraint of the anode bio-electrochemical reactions are needed to complement the reduction of ohmic resistance in modern designs.     

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

Nanoparticle-based detection technology for DNA analysis

With the current rapid development of nanotechnology and synthesis technology for designed oligonucleotides or oligonucleotide-modified nanoparticle conjugates, the combined strategies have become one of the most valuable methods in detection technology for DNA analysis. Using the uniquely recognizable interactions of pre-designed DNA molecules in assembling nanoparticles, various novel approaches have been recently developed towards detecting specific DNA sequences. Here we describe the key fundamentals and issues of this promising strategies ranging from the initial findings of rationally designed DNA-based assembly of nanoparticles to the extended chip-based detection system. Some limitations of these new strategies and possible approaches will be also discussed for the practical application in the area of DNA microarray detection.     

Dual fluorescence nanoconjugates for ratiometric detection of reactive oxygen species in inflammatory cells

Reactive oxygen species (ROS) are largely produced under pathological situations. To understand the etiology of disease, it is urgent to develop efficacious probes for detecting ROS. Herein, a novel nanoconjugate detection system constructed from gold clusters (AuNCs) and quantum dots (QDs) for fluorescence ratiometric‐sensing ROS was reported. Upon interacting with ROS, the red emission fluorescence (645 nm from QDs) in the detection system gradually decreased, while the green fluorescence (480 nm from AuNCs) changed little. The fluorescence ratio at the 2 wavelengths (I_{480 nm}/I_{645 nm}) was linearly correlated with the ROS, which could be used for the real‐time ratiometric detection of ROS. The developed nanoconjugates could be applied to monitor the ROS in inflammatory cells for its ability of generating abundant ROS and uptaking ability to nanoparticles. The stimulated ROS in inflammatory cells were monitored by AuNC‐QD and the results were consistent with the traditional 2′, 7′‐dichlorofluorescin diacetate method, confirming the reliability of the developed method. Featured with the merits of higher photostability, low background, high accuracy of ratiometric detection, the AuNC‐QD conjugate demonstrated its potential to be the probe for real‐time ROS detection in inflammatory cells.