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.     

Automated module for hybridization and staining of commercially produced nucleic acid microarrays

Nucleic acid microarrays are a rapidly expanding technology that enables the detection of pathogens at the genetic level. Currently, the processing of commercially produced microarrays requires cumbersome, expensive, and time-consuming benchtop equipment, which is not practical for point-of-care diagnostic applications. We demonstrate a portable module that can perform the hybridization, wash, and stain steps required for processing a nucleic acid microarray; and it performs these steps in a timeline significantly shorter than the standard commercial protocol. This device is automated, has a small footprint, and serves as a replacement for two commercial pieces of benchtop equipment. Furthermore, our device is designed to serve as a module in a portable biosensor that performs automated sample preparation and nucleic acid amplification. Results with Affymetrix GeneChips show that our device performs as well as non-portable equipment specifically manufactured to process these microarrays. J.S. Erickson and J.E. Hu contributed equally to this work.     

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.     

太赫兹波段超材料在核酸恒温指数扩增检测上的应用

超材料对电磁场具有介电环境敏感和局域电场增强等奇特的电磁特性,近年来广泛用于无标记生物检测。文中设计并制作了一种金属开口谐振环阵列结构的超材料,在太赫兹波段下分别检测核酸恒温指数扩增前后反应体系,在氮气干燥环境下,扩增反应前后的频率偏移量分别为Δf1=（54±3）GHz、Δf2=（60±5）GHz。实验结果显示金属开口谐振环阵列结构的超材料可以作为一种生物传感器快速无标记检测核酸扩增前后的变化。     

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.     

基于信号扩增的荧光技术检测 MicroRNAs 的研究进展

基于信号扩增的荧光技术结合了荧光检测与扩增技术两者的优点,是一种拥有广阔发展前景的生化分析 方法。因其具有扩增效率高、信号易于读取和实时分析等优点,正成为核酸检测中的一个研究热点。文中综述了近年来基于信号扩增的荧光技术应用于 microRNAs 检测的最新进展,包括链置换扩增、滚环扩增、基于外切酶的信号扩增、基于双链特异性核酸酶的信号扩增和无酶信号扩增等,并对今后的发展方向进行了展望。     

Padlock probe-mediated qRT-PCR for DNA computing answer determination

Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (C_t value) and target content. The C_t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.     

A DNA methylation assay for detection of ovarian cancer cells using a HpaII/MspI digestion-based PCR assay in an integrated microfluidic system

Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10² cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.     

DNA计算中核酸序列设计方法比较研究(英文)

DNA计算是将现实问题进行编码,映射到DNA分子上,然后通过分子生物实验产生出代表问题解的DNA分子,最后通过检测技术提取出该DNA分子．高质量的DNA编码可以尽可能避免或减少计算过程中出现的错误,并使检测阶段易于提取出代表问题解的DNA分子．文中对基于汉明距离和基于自由能的DNA核酸编码方法进行研究,分析了两类方法的约束条件对DNA编码质量的影响,比较了两类方法排除非特异性杂交的完备性和计算量,进一步分析了两类方法编码DNA序列的效率．通过分析和比较得到,两类DNA计算编码方法都能有效地限制DNA分子间的非特异性杂交,其中基于汉明距离的DNA编码方法的计算量比较小,但是它仅能近似地估计DNA分子间杂交的热力学稳定性,不能完全替代最小自由能的编码方法．在满足DNA计算试验精度要求的条件下,采用基于汉明距离的DNA编码设计方法不仅能有效地的挑选出特异性杂交和非特异性杂交的DNA序列,还能有效地减少计算量,从而提高DNA序列设计的效率．     

A two-stage electrophoretic microfluidic device for nucleic acid collection and enrichment

Sample purification and enrichment is an important and usually time-consuming step for on-chip nucleic acid detection and analysis. This paper presents an electrophoretic DNA focusing method in microfluidic devices to enrich nucleic acid concentration by around 2700-fold. The electrical waveforms applied to five individual electrodes are such designed that DNAs move successively to the collection electrodes at high speed, while the interferences from bubbles due to electrohydrolysis are minimized. In a spiral channel with a total length of 48 cm, 1 ml DNA sample is purified and enriched by 57 times at a flow rate of 30 μl/min at first. The captured DNAs are then released and transported to the second microfluidic chamber where DNAs are collected and concentrated by 49 times. Thus, in about 40 min, the two-stage device can extract DNAs from 1 ml sample volume and enrich its concentration by 2790-fold. A trade-off exists between the process throughput and the DNA collection efficiency. A DNA capture efficiency of 99.7 % is reached when the flow rate is 1 μl/min, and the maximum DNA capture throughput is achieved at a flow rate of 30 μl/min. As a platform technology, the device can be integrated into bio-sensing and genetic analysis assays for DNA extraction and pre-concentration.     

Multi-functional biochip for medical diagnostics and pathogen detection

We describe a multi-functional biochip (MFB), which uses two different types of bioreceptors, including nucleic acid and antibody probes, on a single platform. The multi-functional capability of the MFB device for biomedical diagnostics is illustrated by measurements of DNA probes specific to gene fragments of Bacillus anthracis and antibody probes targeted to Escherichia coli. Calibration curves for monitoring pathogenic species using antibody probes against E. coli and DNA probes for B. anthracis illustrate the capability of the device for medical diagnostics and for quantitative detection of pathogenic agents.     

A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection

In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wild type. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of interest to decentralized diagnostic platforms.     

DNA一级序列不同编码方法和相似度比较

为了DNA一级序列的相似度计算,本文比较了三种编码方案:单一碱基在DNA序列中的相对位置、二联码即相邻二碱基在序列中的相对位置、编序单一碱基在DNA序列中的相对位置和二联码在序列中的编序相对位置,在此基础上,运用分子连接性指数计算得到序列的不变量,进而,由塔尼莫特法计算得到物种间的相似度。由单一碱基在DNA序列中的相对位置法比较相似度,对于本研究中10个物种,得到了与生物进化树非常相一致的结果。     

Frequency-dependent conductance change of dielectrophoretic-trapped DNA-labeled microbeads and its application in DNA size determinations

DNA amplification is essential in several types of molecular biology approaches. A more rapid and easy analysis of amplicons is still required although many analysis methods have been developed. We have recently devised a new DNA detection method, where DNA amplicons are attached to dielectric microbead surfaces, so that their dielectrophoresis (DEP) force on the microbead reverses polarity, from negative to positive. The DNA-labeled microbeads are trapped on a microelectrode by positive DEP, enabling their rapid detection via DEP impedance measurement. In this paper, we report frequency-dependent conductance of DNA-labeled microbeads. To measure the impedance, sweep-frequency voltage was superimposed on fixed-frequency voltage, with the aim of inducing frequency-dependent conformational change of microbead-attached DNA, ultimately resulting in a change in the conductance of DNA-labeled microbeads. Microbeads labeled with DNA of various sizes (142-, 204-, 391-, and 796-bp) were examined. The normalized conductance sharply decreased at a specific frequency; the frequency was higher with larger DNA size, suggesting a potential application of this method in distinguishing DNA targets according to their size. By combining this method with previously devised DNA detection techniques, both the size and amount of target DNA can be determined within 20 min. This approach is easier and more rapid than conventional methods, such as a gel electrophoresis.     

A microfluidic platform for electrical detection of DNA hybridization

Current methods used for detection of DNA hybridization involve the use of DNA microarrays which require overnight incubation times along with bulky and expensive fluorescent scanners. Here, we demonstrate electrical detection of DNA hybridization in an oligonucleotide functionalized microfluidic channel. We use microchannels functionalized with DNA probes integrated with electrodes for measuring conductance across the channel. As beads conjugated with the target DNA passing through the channel are captured on the surface, we are able to electrically detect changes in resistance due to bead capture. Our assay can be completed in less than an hour using less than a microliter of reagent, and has the potential for extensive multiplexing. Such a device can be useful as a handheld platform in a clinical setting where one would need to rapidly genotype a small number of genes rapidly.     

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.     

A fully integrated microdevice for capturing,amplification, and colorimetric detection of foodborne pathogens

Microsystem Technologies - In the present study, we integrated laboratory functionalities of sample preparation, nucleic acid amplification, and colorimetric detection into a microdevice for the...     

Bead-based polymerase chain reaction on a microchip

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15?min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.     

基于“树枝状”信号放大的电化学DNA生物传感器研究

发展了一种基于"树枝状"信号放大的电化学生物传感器用于DNA的检测。该传感器利用两种DNA功能化的纳米金颗粒,通过两次"三明治"杂交,在电极表面形成"树枝"状结构,从而实现DNA的定量检测。首先通过共价交联方法获得巯基DNA1和DNA2修饰的两种纳米金颗粒,其中DNA1和DNA2与目标cDNA部分互补。然后,修饰在金电极上的捕获探针DNA1与目标cDNA分子及巯基DNA2修饰的纳米金颗粒(DNA2-AuNPs)形成第一个"三明治"杂交结构,实现一次放大检测。接着,DNA2-AuNPs又可与cDNA、巯基DNA1修饰的纳米金颗粒(DNA1-AuNPs)形成第二个"三明治"杂交结构,实现二次放大检测。这种"树枝状"放大信号的方法的检测限是0.13pmol/L,相对仅利用纳米金颗粒放大的方法而言,其检测限降低了4倍。并且,该传感器具有较好的识别碱基错配的能力。