Microfluidic devices harboring unsealed reactors for real-time isothermal helicase-dependent amplification

High-throughput microchip devices used for nucleic-acid amplification require sealed reactors. This is to prevent evaporative loss of the amplification mixture and cross-contamination, which may occur among fluidically connected reactors. In most high-throughput nucleic-acid amplification devices, reactor sealing is achieved by microvalves. Additionally, these devices require micropumps to distribute amplification mixture into an array of reactors, thereby increasing the device cost, and adding complexity to the chip fabrication and operation processes. To overcome these limitations, we report microfluidic devices harboring open (unsealed) reactors in conjunction with a single-step capillary based flow scheme for sequential distribution of amplification mixture into an array of reactors. Concern about evaporative loss in unsealed reactors have been addressed by optimized reactor design, smooth internal reactor surfaces, and incorporation of a localized heating scheme for the reactors, in which isothermal, real-time helicase-dependent amplification (HDA) was performed.     

Compact optical diagnostic device for isothermal nucleic acids amplification

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R² = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R² = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h.     

Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller

Fabrication of 3D microfluidic devices is normally quite expensive and tedious. A strategy was established to rapidly and effectively produce multilayer 3D microfluidic chips which are made of two layers of poly(methyl methacrylate) (PMMA) sheets and three layers of double-sided pressure sensitive adhesive (PSA) tapes. The channel structures were cut in each layer by cutting plotter before assembly. The structured channels were covered by a PMMA sheet on top and a PMMA carrier which contained threads to connect with tubing. A large variety of PMMA slides and PSA tapes can easily be designed and cut with the help of a cutting plotter. The microfluidic chip was manually assembled by a simple lamination process.The complete fabrication process from device design concept to working device can be completed in minutes without the need of expensive equipment such as laser, thermal lamination, and cleanroom. This rapid frabrication method was applied for design of a 3D hydrodynamic focusing device for synthesis of gold nanoparticles (AuNPs) as proof-of-concept. The fouling of AuNPs was prevented by means of a sheath flow. Different parameters such as flow rate and concentration of reagents were controlled to achieve AuNPs of various sizes. The sheet-based fabrication method offers a possibility to create complex microfluidic devices in a rapid, cheap and easy way.

     

Sub-microliter scale in-gel loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a common human disease that is prevalent in resource-deprived areas of the world. Current detection techniques for TB require expensive conventional instruments in a laboratory setting, preventing accessible and low cost diagnosis of the disease. Using a loop-mediated isothermal amplification (LAMP) assay, we have amplified and detected TB in a 6 × 8 semisolid polyacrylamide gel post array using an inexpensive prototype instrument. Each post contains 670 nL of volume, minimizing the need for large quantities of reagents. Amplified DNA is detected via fluorescence of the dye LCGreen Plus+, which is polymerized into the gel along with other reagents. The prototype device contains a Peltier element for heating, a diode laser as an excitation source, and a CCD camera for detecting fluorescence in real-time. About 12 Mycobacterium tuberculosis genomes per gel post can be detected within 75 min of amplification. This sensitivity is similar to that obtained by conventional methods using a commercial thermocycler. We achieved comparable LAMP amplification when the template is added externally or when the template is polymerized in the gel. This rapid isothermal amplification technology, with its simple thermal requirements, has the potential to be integrated into micro-devices and serves as a model for implementing future low-cost point of care diagnostics.     

A disposable,integrated loop-mediated isothermal amplification cassette with thermally actuated valves

An inexpensive, disposable, integrated, polymer-based cassette for loop-mediated isothermal amplification (LAMP) of target nucleic acids was designed, fabricated, and tested. The LAMP chamber was equipped with single-use, thermally actuated valves made with a composite consisting of a mixture of PDMS and expandable microspheres. The effect of the composite composition on its expansion was investigated, and the valve’s performance was evaluated. In its closed state, the valve can hold pressures as high as 200 kPa without any significant leakage. Both the LAMP chamber and the valves were actuated with thin film heaters. The utility of the cassette was demonstrated by carrying out LAMP of Escherichia coli DNA target and reverse transcribed loop meditated isothermal amplification (RT-LAMP) of RNA targets. The amplicons were detected in real time with a portable, compact detector. The system was capable of detecting as few as 10 target molecules per sample in well under 1 h. The portable, integrated cassette system described here is particularly suited for applications at the point of care and in resource-poor countries, where funds and trained personnel are in short supply.     

Miniaturized Vision System for Microfluidic Devices

One of the biggest obstacles for lab-on-chip (LOC) devices is the miniaturization of large-scale devices and its methodologies. Miniaturization of the current microscopic technologies combined with image processing may bring significant advantages for LOC devices in the dynamic processes of sizing, positioning, flow control and cell manipulation at different time scales. Here, we propose a vision system boarded on a polydimethylsiloxane (PDMS) polymer-based chip, which can be utilized in a complex microfluidic network for continuous monitoring of mammalian egg and donor cells of sizes in the range of 10–100 μm. The developed prototype system has sufficient resolution and is accompanied with a robust detection method for cell-based microfluidic applications. To assess its performance, an image processing algorithm was applied, and the capability of the detection method was evaluated using 11- and 26-μm particles. The results show that the proposed optical system of monitoring and illumination can be effectively incorporated into PDMS structures aiming at LOC devices.     

Generalized addressing schemes for data graphs

Addressable data graphs enjoy structural properties which are at once mathematically attractive and practically useful. This paper is devoted to studying generalizations of addressability, with a twofold goal. The first aim of the paper is to gain understanding of what features of addressing schemes account for the attractive properties of addressable data graphs. The second purpose of the paper is to seek a variant of the property of addressability, which retains the practical concomitants of the original property, but which is enjoyed by a broader class of data graphs. Two such variants are discovered,quasi-addressability andweak-addressability. Although most data graphs do not enjoy any form of addressability, every data graph can be slightly modified to render it quasi or weakly addressable—in sharp contrast to the stringent demands of addressability. However, the added breadth of these new addressabilities is obtained at the expense of the invariance and selectivity which accompany the original property.     

Autonomous programmable DNA nanorobotic devices using DNAzymes

A major challenge in nanoscience is the design of synthetic molecular devices that run autonomously (that is, without externally mediated changes per work-cycle) and are programmable (that is, their behavior can be modified without complete redesign of the device). DNA-based synthetic molecular devices have the advantage of being relatively simple to design and engineer, due to the predictable secondary structure of DNA nanostructures and the well-established biochemistry used to manipulate DNA nanostructures. However, ideally we would like to minimize the use of protein enzymes in the design of a DNA-based synthetic molecular device. We present the design of a class of DNA-based molecular devices using DNAzyme. These DNAzyme-based devices are autonomous, programmable, and further require no protein enzymes. The basic principle involved is inspired by a simple but ingenious molecular device due to Tian et al. [Y. Tian, Y. He, Y. Chen, P. Yin, C. Mao, A DNAzyme that walks processively and autonomously along a one-dimensional track, Angew. Chem. Intl. Ed. 44 (2005) 4355–4358] that used DNAzyme to traverse on a DNA nanostructure, but was not programmable in the sense defined above (it did not execute computations).     

Off-lattice simulation of the solid phase DNA amplification

Recent simulations of the solid phase DNA amplification (SPA) by J.-F. Mercier et al. [Biophys. J. 85 (2003) 2075] are generalized to include two kinds of primers and the off-lattice character of the primer distribution on the surface. The sigmoidal character of the primer occupation by DNA, observed experimentally, is reproduced in the simulation. We discuss an influence of two parameters on the efficiency of the amplification process: the initial density p₀ of the occupied primers from the interfacial amplification and the ratio r of the molecule length to the average distance between primers. The number of cycles required for a complete saturation decreases with p₀ roughly as . For r=1.5, the number of occupied primers is reduced by a factor two, when compared to the case of longer molecules. Below r=1.4, the effectivity of SPA is reduced by a factor 100.     

基于核酸识体的滚环DNA扩增反应用于PDGF-BB的检测

该文提出了一种新颖的、高灵敏的基于抗体-抗原-核酸识体夹心法和DNA滚环扩增(RCA)的方法用于血小板源生长因子BB(PDGF-BB)的检测.在核酸识体的3'端连接RCA反应的引物,在φ29 DNA聚合酶、脱氧核糖核苷酸及环形模板存在下,经过滚环扩增反应后,可得到一条与环形DNA模板互补的重复序列的DNA单链,利用生物素标记的互补寡核苷酸探针与扩增产物杂交,再用亲和素标记的碱性磷酸酶与之反应,将反应后的产物在底物溶液中进行银沉积反应,最后用电化学方法检测电极表面沉积的银量,通过检测银量来达到检测PDGF-BB的目的.考察了抗体固定化浓度、RCA反应时间和其它蛋白质对测定信号的影响.结果表明,RCA扩增反应能显著的提高检测的灵敏度,降低检测下限.PDGF-BB在0.5 pg/mL～8 000 pg/mL范围内呈良好的线性关系,检测下限为0.2 pg/mL.该方法在蛋白质的检测和医学诊断等方面有很大的应用前景.     

Miniaturized plastic micro plates for applications in HTS

This paper is focused on the development of plastic nano titer plates for applications in high throughput screening (HTS). For screening systems with integrated confocal microscopes plastic chips have been fabricated by injection molding and injection compression molding which contain micro wells with volumes of 0.9 μl and 1.4 μl and bottom plates with thicknesses of 120 μm and 200 μm. In addition, plastic chips with through holes have been joined with 160 μm glass plates by an adhesive printing process. First fluorescence correlation spectroscopy (FCS) measurements show that the plastic plates with glass bottoms are qualified as screening grade FCS nano titer plates. Received: 15 May 1999 / Accepted: 19 May 1999     

Field interactions and distinctive features of development of devices for diagnostics and correction of states of biologically active points

Based on a model of field interactions of thermochemical potentials, the possibility of correlation between the conductance and other parameters of biologically active points (BAPs) is investigated. New methods are described for diagnostics and correction of physiological systems by mild actions on BAPs. These methods can be taken into account in developing new devices for reflexotherapy. __________ Translated from Kibernetika i Sistemnyi Analiz, No. 2, pp. 157–165, March–April 2006.     

Microfabrication of single-use plastic microfluidic devices for high-throughput screening and DNA analysis

 Modern drug discovery and genomic analysis depend on rapid analysis of large numbers of samples in parallel. The applicability of microfluidic devices in this field needs low cost devices, which can be fabricated in mass production. In close collaboration, Greiner Bio-One and Forschungszentrum Karlsruhe have developed a single-use plastic microfluidic capillary electrophoresis (CE) array in the standardized microplate footprint. Feasibility studies have shown that hot embossing with a mechanical micromachined molding tool is the appropriate technology for low cost mass fabrication. A subsequent sealing of the microchannels allows sub-microliter sample volumes in 96-channel multiplexed microstructures. Received: 16 May 2001 / Accepted: 3 July 2001     

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

The design of autonomous DNA nano-mechanical devices: Walking and rolling DNA

We provide designs for the first autonomousDNA nanomechanical devices that execute cycles of motion without external environmental changes. These DNA devices translate along a circular strand of ssDNA and rotate simultaneously. The designs use various energy sources to fuel the movements, include (i) ATP consumption by DNA ligase in conjunction with restriction enzyme operations, (ii) DNA hybridization energy in trapped states, and (iii) kinetic (heat) energy. We show that each of these energy sources can be used to fuel random bidirectional movements that acquire after n steps an expected translational deviation of O(√n). For the devices using the first two fuel sources, the rate of stepping is accelerated over the rate of random drift due to kinetic (heat) energy. Our first DNA device, which we call walking DNA, achieves random bidirectional motion around a circular ssDNA strand by use of DNA ligase and two restriction enzymes. Our other DNA device, which we call rolling DNA, achieves random bidirectional motion without use of DNA ligase or any restriction enzyme, and instead using hybridization energy. We also describe how to modify the design for the rolling DNA device to include a ``latching mechanism' that fixes the wheels position at specified location along the road, so as to provide for overall unidirectional translational movement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Miniaturized fluorescence excitation platform with optical fiber for bio-detection chips

This paper presents a new research study on the fabrication of a fluorescence bio-detection chip with an optical fiber transmission platform. Anisotropic wet etching on (100) silicon wafers to fabrication V-groove for optical fiber alignment and micro-mirror were included. Combined with anodic bonding technique to adhere glass, silicon structure and optical fiber for a fluorescence excitation platform was completed. In this study, a 40% KOH etching solution was used to study the parameter effect. The results show that the working temperature is the main parameter that significantly effects the etch rate. The anisotropic etching resulted in 54.7° reflective mirrors and the reflectivity for the optical beam was also examined. The surface roughness of the micro-mirror is Ra 4.1 nm measured using AFM providing excellent optical reflection. The incident light and beam profiles were also examined for further study. This study shows that this micro-platform is adaptable for fluorescence bio-detection.     

Miniaturized octupole cytometry for cell type independent trapping and analysis

Microflow cytometry, including robust alignment, separation, and trapping of living cells, is on the verge of commercialization. Yet, the necessary equipment is frequently not applicable to certain biological questions as the products have been specifically developed for particular cell types. We present a versatile cell handling technology based on single miniaturized octupoles that enables the physical manipulation of a broad variety of different cell types via controlled negative dielectrophoresis force fields. The octupole technology allows contactless and time-resolved cell analysis in physicochemical controlled microenvironments. Contactless cell manipulation and trapping with the octupole technology were shown to be independent of cell size and morphology. This was demonstrated with nine different cell types of technical and medical relevance, ranging from motile bacteria over yeast and small platelets (thrombocytes) up to large cancer cells. We also demonstrate applications of octupole cytometry for controlled analyses of mechano-elastic properties of single cells, contactless cultivation and perfusion for perturbation studies, as well as studying the interaction of different cell types in physical proximity. These examples prove the miniaturized octupole format as a versatile, noninvasive, and robust tool for microfluidic single cell cytometry that complements existing hydrodynamic, optical, and acoustic technologies.