3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers

Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.     

Giant magnetoresistive biochip for DNA detection and HPV genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.     

Detection of multi-color fluorescent objects with single photon spectrometer

Single photon counting is the most sensitive and accurate method for detection of very weak fluorescent signals obtained in many applications such as DNA sequencing, detection of biological reporters on micro-beads, detection of droplets in micro-fluidic systems, etc. In this paper we describe the use of single photon spectrometer for detection and characterization of very weak multicolor fluorescence produced by mixtures of various fluorescent dyes and quantum dots.     

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.     

Nanoparticle interactions with blood proteins and what it means: a tutorial review

Nanoparticles find many uses in medicine and biomedical technology. Such applications imply that they must be colloidally stable and do not interact with proteins in the blood or blood serum. A nanoparticle put into the blood will instantaneously be covered by a protein corona that compromises the function of the nanoparticle core, changes the effective size of the nanoparticle, and determines its biological fate. Strategies developed to gain control over nanoparticles in biological fluids, particular in blood, heavily rely on creating a hydrated polymer shell that sterically and osmotically prevents a protein corona from forming. In this tutorial review, we provide an overview of factors that affect the formation of the protein corona in blood and how to prevent it forming. We focus on describing the latest advances in our understanding of how small core-shell nanoparticles (core diameter 4-20 nm in diameter) with a shell of densely grafted polymer chains, a so-called polymer brush, interact with proteins and cells in vitro. Such nanoparticles are among the most well-defined and well-characterized colloids used for biomedical applications, from which an improved understanding of how nanoparticle architecture influences their biological fate can be obtained in detail.     

Detection of uncharged or feebly charged small molecules by field-effect transistor biosensors

This paper describes a new technique for the detection of uncharged or feebly charged small molecules (<400Da) using Si field-effect transistor (FET) biosensors that are signal-enhanced by gold nanoparticle (NP) charges under dry measurement conditions. NP charges are quickly induced by a chemical deposition (that is, Au deposition) and the indirect competitive immunogold assay, and strongly enhance the electrical signals of the FET biosensors. For the validation of signal enhancement of FET biosensors based on NP charges and detection of uncharged or feebly charged small molecules, mycotoxins (MTXs) of aflatoxin-B1 (AFB1), zearalenone (ZEN), and ochratoxin-A (OTA) were used as target molecules. According to our experimental results, the signal is 100 times more enhanced than the use of the existing solution FET biosensing techniques. Furthermore, this method enables the FET biosensor to quantitatively detect target molecules, regardless of the ionic strengths, isoelectric points (pI), or pHs of the measured sample solutions.     

Mathematical modelling and computer simulation of Brownian motion and hybridisation of nanoparticle–bioprobe–polymer complexes in the low concentration limit

A wide variety of nano-biotechnological applications are being developed for nanoparticles based on in vitro diagnostic and imaging systems. Some of these systems allow highly sensitive detection of molecular biomarkers. Frequently, the very low concentration of the biomarkers makes very difficult the mathematical simulation of the motion of nanoparticles based on classical, partial differential equations. We address the issue of incubation times for low concentration systems using Monte Carlo simulations. We describe a mathematical model and computer simulation of Brownian motion of nanoparticle–bioprobe–polymer contrast agent complexes and their hybridisation to immobilised targets. We present results for the dependence of incubation times on the number of particles available for detection, and the geometric layout of the DNA-detection assay on the chip.     

The linear electrode array: a useful tool with many applications.

In this review we describe the basic principles of operation of linear electrode arrays for the detection of surface EMG signals, together with their most relevant current applications. A linear array of electrodes is a system which detects surface EMG signals in a number of points located along a line. A spatial filter is usually placed in each point for signal detection, so that the recording of EMG signals with linear arrays corresponds to the sampling in one spatial direction of a spatially filtered version of the potential distribution over the skin. Linear arrays provide indications on motor unit (MU) anatomical properties, such as the locations of the innervation zones and tendons, and the fiber length. Such systems allow the investigation of the properties of the volume conductor and its effect on surface detected signals. Moreover, linear arrays allow to estimate muscle fiber conduction velocity with a very low standard deviation of estimation (of the order of 0.1-0.2 m/s), thus providing reliable indications on muscle fiber membrane properties and their changes in time (for example with fatigue or during treatment). Conduction velocity can be estimated from a signal epoch (global estimate) or at the single MU level. In the latter case, MU action potentials are identified from the interference EMG signals and conduction velocity is estimated for each detected potential. In this way it is possible, in certain conditions, to investigate single MU control and conduction properties with a completely non-invasive approach. Linear arrays provide valuable information on the neuromuscular system properties and appear to be promising tools for applied studies and clinical research.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

基于节律性脑电信号的脑-机接口

脑-机接口系统是一个不依靠外周神经和肌肉组织等而实现大脑和外界装置之间直接的交流和控制的通道。它为那些运动障碍的残疾人表达自己的意愿和实现对外部设备的控制提供了一种新的强大的技术支持。基于脑电的脑-机接口作为一种非侵入型的技术引起了该领域很多人的关注。基于脑电的脑-机接口采用了很多种类型的脑电信号。其中,振荡性的脑电图由于有较高的幅值和对噪声不敏感等特性而体现出极大的优势。也是由于这些原因,振荡性的脑电图变成了脑-机接口的应用中非常成功的设计之一。本文要介绍主要的基于脑电的脑-机接口中的两种,分别是稳态视觉诱发电位和基于运动本体感觉节律的脑-机接口。作者将详细的叙述该研究的生理背景、脑-机接口的参数,以及该系统的构造及信号处理的方法,并且会演示一些具有潜在应用价值的科研成果。     

Nanoparticles: potential biomarker harvesters

A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.     

Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review

Hydrogels are physically or chemically cross-linked polymer networks that are able to absorb large amounts of water. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. The present review aims to give an overview of hydrogels based on natural polymers and their various applications in the field of tissue engineering. In a first part, relevant parameters describing different hydrogel properties and the strategies applied to finetune these characteristics will be described. In a second part, an important class of biopolymers that possess thermosensitive properties (UCST or LCST behavior) will be discussed. Another part of the review will be devoted to the application of cryogels. Finally, the most relevant biopolymer-based hydrogel systems, the different methods of preparation, as well as an in depth overview of the applications in the field of tissue engineering will be given.     

The use of nanocrystals in biological detection

In the coming decade, the ability to sense and detect the state of biological systems and living organisms optically, electrically and magnetically will be radically transformed by developments in materials physics and chemistry. The emerging ability to control the patterns of matter on the nanometer length scale can be expected to lead to entirely new types of biological sensors. These new systems will be capable of sensing at the single-molecule level in living cells, and capable of parallel integration for detection of multiple signals, enabling a diversity of simultaneous experiments, as well as better crosschecks and controls.     

Exploring Age-Related Changes in Dynamical Non-Stationarity in Electroencephalographic Signals during Early Adolescence

Dynamics of brain signals such as electroencephalogram (EEG) can be characterized as a sequence of quasi-stable patterns. Such patterns in the brain signals can be associated with coordinated neural oscillations, which can be modeled by non-linear systems. Further, these patterns can be quantified through dynamical non-stationarity based on detection of qualitative changes in the state of the systems underlying the observed brain signals. This study explored age-related changes in dynamical non-stationarity of the brain signals recorded at rest, longitudinally with 128-channel EEG during early adolescence (10 to 13 years of age, 56 participants). Dynamical non-stationarity was analyzed based on segmentation of the time series with subsequent grouping of the segments into clusters with similar dynamics. Age-related changes in dynamical non-stationarity were described in terms of the number of stationary states and the duration of the stationary segments. We found that the EEG signal became more non-stationary with age. Specifically, the number of states increased whereas the mean duration of the stationary segment decreased with age. These two effects had global and parieto-occipital distribution, respectively, with the later effect being most dominant in the alpha (around 10 Hz) frequency band.     

Nanoparticles containing insoluble drug for cancer therapy

Nanoparticle drug formulations have been extensively researched and developed in the field of drug delivery as a means to efficiently deliver insoluble drugs to tumor cells. By mechanisms of the enhanced permeability and retention effect, nanoparticle drug formulations are capable of greatly enhancing the safety, pharmacokinetic profiles and bioavailability of the administered treatment. Here, the progress of various nanoparticle formulations in both research and clinical applications is detailed with a focus on the development of drug/gene delivery systems. Specifically, the unique advantages and disadvantages of polymeric nanoparticles, liposomes, solid lipid nanoparticles, nanocrystals and lipid-coated nanoparticles for targeted drug delivery will be investigated in detail.     

Enhancing ATP-based bacteria and biofilm detection by enzymatic pyrophosphate regeneration

The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PP_i in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5′-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PP_i and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines.     

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.     

生物分子的纳米粒子标记和检测技术

生物分子的标记和检测一直是生物分析领域的重要内容 .近年来 ,纳米材料与生物检测技术的结合 ,使得生物分子的检测有了重要的发展 ,这一交叉学科现已成为生物分析领域最具活力的研究方向 .对近期出现的新型纳米粒子标记物的性质、检测原理、特点和应用进行了评述 ,并分析了用该标记物进行分析的可能发展方向     

Online optical detection of food contaminants in microdroplets

To accommodate the considerable increase of disease based on microbial food contaminants in the last decade, a modulated, fast optical fluorescence detection combined with microdevices is created. This method, which consists of five different steps, first selects contaminants, mainly bacteria, in the food matrix. This process is based on a biomagnetic separation technique developed by our collaborators at the Technical University of Dresden. By the steps of binding antibody functionalized magnetic beads and fluorescent capsules on the target cell, a magnetic bead‐target cell‐microcapsule complex (MTM) is generated. The well‐established pipe‐based bioreactors (pbb) platform enables the generation of droplets with a volume between 60 and 160 nL and the detection of the target cell with an integrated microscopic and spectroscopic detection system. The module used for generating droplets is based on the segmented flow principle and is chip‐ or probe‐based. In this context, the successful use of polydimethylsiloxane (PDMS) as a cost‐effective alternative to the well‐established glass‐chips is introduced. To quantify the detection based on a yes‐ or no‐decision, the most important step is to separate one MTM‐complex per droplet. This equalized the quantity of the fluorescent signals with the quantity of the contaminants in the cell sample. The feasibility of microscopic and spectroscopic detection with only one fluorescent capsule per droplet is shown. Also the first results of a special prototyping optical detection set‐up that is already in an advanced stage of development, will be presented. This easy‐to‐use device implemented a software‐controlled, automatic documentation for every fluorescent signal of a droplet to guarantee the quality control. Here are the advantages of an integration of microdevices in a rapid detection of food pathogens presented. Obviously, the modular set‐up of this detection platform enables a wide range of high‐throughput applications.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.