Label-free detection of DNA hybridization using gold-coated tapered fiber optic biosensors (TFOBS) in a flow cell at 1310 nm and 1550 nm

We had previously reported the detection of a model protein bovine serum albumin (BSA) using antibody-immobilized tapered fiber optic biosensors (TFOBS) at 1310 nm and 1550 nm under stagnant and flow conditions. Because of recent interest in pathogen detection based on DNA, in this work we explore the application of these sensors for the detection of single stranded DNA (ssDNA). We show that it is feasible to directly detect the hybridization of a 10-mer ssDNA to its complementary strand immobilized on the sensor surface. Detection was performed under flow conditions because flow reduces non-specific binding to sensor surface, eliminates optical transmission changes due to mechanical movements, and allows for instantaneous switching of samples when needed.

TFOBS were fabricated with waist diameters of 5–10 μm and total lengths of 1000–1200 μm. The taper regions were coated with 50 nm of gold and housed in a specially constructed holder which served as a flow cell. The TFOBS was immobilized with 15-mer ssDNA with a C₆ extension and a thiol group, which attaches to Au1 1 1 sites. Then, the complementary 10-mer ssDNA samples were allowed to flow in from low to high concentration (750 fM to 7.5 nM) and the resulting transmission changes were recorded. It is shown that 750 fM of complementary DNA can be detected. This sensor was able to distinguish between complementary DNA from DNA with a single nucleotide mismatch in the middle position.     

Sensitive PAT gene sequence detection by nano-SiO2/p-aminothiophenol self-assembled films DNA electrochemical biosensor based on impedance measurement

Nano-SiO₂/p-aminothiophenol (PATP) film was fabricated by self-assembly and electrodeposition methods. The immobilization and hybridization of DNA on the nano-SiO₂/PATP film were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was applied to label-free detection of the target DNA according to the increase of the electron transfer resistance (R_et) of the electrode surface after the hybridization of the probe DNA with the target DNA. This DNA electrochemical biosensor showed its own performance of simplicity, good stability, fine selectivity and high sensitivity, and was successfully applied to the detection of the PAT gene sequences by a label-free EIS method. The dynamic detection range was from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁶ mol/L 20-base sequence of the PAT gene, with the detection limit of 1.5 × 10⁻¹² mol/L. This DNA sensor has a good ability of recognizing single- or double-base mismatched DNA sequence with the complementary DNA sequence.     

Dispersion of single-walled carbon nanotubes in poly(diallyldimethylammonium chloride) for preparation of a glucose biosensor

Poly(diallyldimethylammonium chloride) (PDDA) was chosen to disperse single-walled carbon nanotubes (SWCNTs). The optimal conditions to prepare stable PDDA–SWCNTs aqueous dispersions were presented. Then, the positively charged PDDA–SWCNTs composite and negatively charged glucose oxidase (GOD) were employed to fabricate multilayer films on platinum (Pt) electrodes by layer-by-layer self-assembly technique. The consecutive growth of the multilayer films was confirmed by quartz crystal microbalance. Electrochemical measurements were used to study the properties of the proposed biosensor. Results demonstrated that SWCNTs were evenly dispersed within the PDDA films and efficiently improved the conductivity of the resulting films. Among the biosensors, the one based on seven layers of multilayer films got the best performance. It showed wide linear range of 0.05–12 mM, high sensitivity of 63.84 μA/(mM cm²), low detection limit of about 4 μM and small value of the apparent Michaelis–Menten constant, 8.46 mM. In addition, the biosensor also exhibited good suppression of interference and long-term operational stability. This protocol could be used to immobilize other enzymes to construct a range of biosensors.     

Development of an SH-SAW sensor for the detection of DNA hybridization

We have developed SH (shear horizontal) surface acoustic wave (SAW) sensors for the detection of DNA (deoxyribonucleic acid) hybridization on the gold-coated delay line of transverse SAW devices. DNA hybridization experiments were performed with 15-mer oligonucleotides (probe and complementary target DNA). The sensor consists of twin SAW delay line oscillators (sensing channel and reference channel) operating at 100 MHz fabricated on 36° rotated Y-cut X-propagation LiTaO₃ piezoelectric single crystals. The relative change in the frequency of the two oscillators was monitored to detect hybridization between the target DNA and probe DNA immobilized on the delay line of the SAW sensor. The measurement results showed a good response of the sensor to the mass loading effects of the DNA hybridization with a sensitivity level up to 1.55 ng/ml/Hz.     

一种电流积分型生物传感微阵列信号读出电路

设计了一种用于自组装膜生物传感阵列的高灵敏度信号读出电路,该电路主要包括高灵敏度微阵列生物电流探测单元、积分单元、相关双采样(CDS)单元及输出缓冲单元。电路采用单5 V电源,输入电流为0~50 nA,在0.6μm/level 7 CMOS工艺条件下进行模拟,得到了较为满意的结果。该读出电路与标准CMOS工艺兼容,可实现集成的生物传感阵列。     

一种用于集成电化学生物传感阵列的高灵敏度 读出电路的研究

一种用于自组装膜生物传感阵列的高灵敏度信号读出电路包含一个高灵敏度电流读出放大器,可以接受pA-nA范围的输入电流,其电流增益大约60 dB,相位裕度为63°,并在同一输入电流信号的条件下,负载在一个较大的范围变化时,其输出电流几乎几乎是一个常数.芯片采用1.2μm CMOS工艺加工,测试结果表明,该芯片的输出电流对输入电流具有较好的线性度.这种新型电路与标准CMOS工艺兼容,可实现集成的生物传感阵列.     

DNA hybridization measurement by self-sensing piezoresistive microcantilevers in CMOS biosensor

Understanding the mechanism of how biological reactions produce mechanical loadings is fundamental to biomedical developments. A CMOS biosensor chip is developed to measure in situ the induced surface stress change by DNA hybridization. For 20-mer thiol-modified single stranded DNA (ssDNA), the mechanism of ssDNA attached to gold surface via a sulfur–gold linkage can be investigated by using the Langmuir adsorption model. Experimental results indicate that the immobilization response is less than 1 s, the total number of ssDNA molecules on the cantilever is about 3 × 10¹¹, and the induced surface stress is 0.15 N/m. The surface stress sensitivity of the sensor is about 3.5 × 10⁻⁵ m/N. The estimated adsorption rate of the ssDNA is 0.005 s⁻¹. The biosensor is capable of discriminating complimentary molecular targets and thus may provide a powerful platform for high throughput real-time analysis of DNA.     

A fully integrated 1‐in. AMOLED display using current feedback based on a five‐mask LTPS CMOS process

Abstract— In the past, a five‐mask LTPS CMOS process requiring only one single ion‐doping step was used. Based on that process, all necessary components for the realization of a fully integrated AMOLED display using a 3T1C current‐feedback pixel circuit has recently been developed. The integrated data driver is based on a newly developed LTPS operational amplifier, which does not require any compensation for Vth or mobility variations. Only one operational amplifier per column is used to perform digital‐to‐analog conversion as well as current control. In order to achieve high‐precision analog behavior, the operational amplifier is embedded in a switched capacitor network. In addition to circuit verification by simulation and analytic analysis, a 1‐in. fully integrated AMOLED demonstrator was successfully built. To the best of the authors' knowledge, this is the first implementation of a fully integrated AMOLED display with current feedback.     

A particle swarm optimization method for fault localization and residue removal in digital microfluidic biochips

Second generation microfluidic biochip is known as digital microfluidic biochip (DMFB). DMFB performs different clinical pathological experimentation, DNA sequencing, air contamination detection and many other bio-chemical experiments based on appropriate bio-assay protocols. DMFB comprises two dimensional array of electrodes fabricated over two parallel glass plates, which is capable of performing miniaturized (nano/ pico liter volume) droplet dispensing, transportation and mixing through electro-wetting of dielectrics (EWOD). Droplet operations through EWOD are mostly managed by droplet scheduling algorithms which are NP hard in nature. At present reliability is a major concern for commercialization of operational DMFB. Reliable output of DMFB is mostly affected by different faults within electrodes. This further suffers from cross-contamination issues among different droplets due to repeated use of DMFB boards for different assay operations. Present work proposes a novel test droplet routing method based on adaptive weighted particle swarm optimization (PSO) model. This test droplet circulation method aims to identify defective electrodes and simultaneously performs residue removal. Experimental findings of the proposed model on some standard test benches and real life bio-assay samples reveal operational supremacy in terms of overall computational time and operational accuracy over some existing best known models.     

On existence of reporter strands in DNA-based graph structures

Through self-assembly of branched junction molecules many different DNA structures (graphs) can be assembled. We show that every multigraph can be assembled by DNA such that there is a single strand that traces each edge in the graph at least once. This strand corresponds to a boundary component of a two-dimensional orientable surface that has the given graph as a deformation retract. This boundary component traverses every edge at least once, and it defines a circular path in the graph that “preserves the graph structure” and traverses each edge.     

可满足问题的分子信标计算模型(英文)

分子信标（Molecular Beacon）是一种发夹状的荧光探针,它可以特异地和那些与分子信标的环（Loop）互补的核酸靶序列杂交,具有单个碱基错配的检测能力．肽核酸（Peptide Nucleic Acid）是人工合成的核酸（DNA）的类似物．PNA骨架为酰胺键,与DNA补链杂交更稳定,可以阻止聚合酶延伸反应．文中将可满足问题的约束变量编码于分子信标的环部识别区,通过分子信标与使得给定范式为真的变量的PNA补链杂交,再利用PNA链可以阻止聚合酶延伸反应的性质,用限制性内切酶EcoRI降解对应于非解的分子信标,最后通过加热表面使分子信标构形发生变化,产生荧光读解．提出的可满足问题的分子信标计算模型具有可靠性高、无需观察和记录计算的中间结果、读解简单等优点．     

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.     

Sensitive electrochemical immunosensor based on enlarged and surface charged gold nanoparticles mediated electron transfer

A simple and sensitive electrochemical immunosensor for the detection of protein biomarker interleukin-6 (IL-6) based on gold nanoparticles (AuNPs) as label was reported. The signal of the immunosensor was originated from enlarged and positively charged AuNPs mediated electron transfer between insulating self-assemble monolayer (SAM) modified electrode and K₃Fe(CN)₆ solution. The gold electrode was first modified with SAM to block the electron transfer between the electrode and K₃Fe(CN)₆ solution. After the preparation of the immunosensor, the AuNPs attached onto electrode was enlarged and positively charged when treated in solution containing HAuCl₄, ascorbic acid and cetyltrimethylammonium bromide (CTAB). The enlarged and positively charged AuNPs then mediated electron transfer between the electrode and K₃Fe(CN)₆ solution, creating redox current that is proportional to the concentration of IL-6 detected. The reported immunosensor has high sensitivity and wide linear range. This novel signal amplification strategy can be applied to the detection of different kinds of protein biomarkers.     

嵌入式生物芯片检测分析系统设计*

提出一种嵌入式生物芯片检测分析系统设计方法。根据样点、噪声和背景特征的关系提出适合嵌入式系统的软件算法,并设计出通过CCD采集芯片图像、DSP进行图像信息处理的生物芯片检测分析系统硬件。多种生物芯片实验结果表明系统操作简便,可以稳定地对生物芯片进行自动的检测分析,对目前生物芯片检测分析系统的自动性和集成性方面有很大的提升。     

Actively recharged single photon counting avalanche photodiode integrated in an industrial CMOS process

An actively recharged single photon counting avalanche photodiode (SPAD) is integrated in a conventional CMOS process. A fast recharge through a low impedance path leads to a dead time lower than 10 ns. This outstanding feature allows one to work with pulse repetition rate up to 100 MHz in time correlated single photon counting based experiments. Biased 2.5 V above its breakdown voltage, the 30 μm² sensitive area photodiode has a maximum detection probability of about 20% at λ=440 nm and up to 5% in the visible part of the spectrum. At this bias condition, the dark count rate is as low as 60 Hz at room temperature, making a cooling of the microsystem unnecessary. The AR-SPAD exhibits no afterpulsing phenomenon revealing the maturity of the CMOS process used. The timing resolution of the AR-SPAD is less than 50 ps. For applications where the photons can be focused on the detector with an objective, the AR-SPAD is highly competitive with commercially available single photon counter. Furthermore, CMOS integration opens the way to arrays fabrication as well as co-integration of additional functions to develop smart optical sensors.     

Automating fluid delivery in a capillary microfluidic device using low-voltage electrowetting valves

A multilayer capillary polymeric microfluidic device integrated with three normally closed electrowetting valves for timed fluidic delivery was developed. The microfluidic channel consisted two flexible layers of poly (ethylene terephthalate) bonded by a pressure-sensitive adhesive spacer tape. Channels were patterned in the spacer tape using laser ablation. Each valve contained two inkjet-printed silver electrodes in series. Capillary flow within the microchannel was stopped at the second electrode which was modified with a hydrophobic monolayer (valve closed). When a potential was applied across the electrodes, the hydrophobic monolayer became hydrophilic and allowed flow to continue (valve opened). The relationship between the actuation voltage, the actuation time, and the distance between two electrodes was performed using a microfluidic chip containing a single microchannel design. The results showed that a low voltage (4.5 V) was able to open the valve within 1 s when the distance between two electrodes was 1 mm. Increased voltages were needed to open the valves when the distance between two electrodes was increased. Additionally, the actuation time required to open the valve increased when voltage was decreased. A multichannel device was fabricated to demonstrate timed fluid delivery between three solutions. Our electrowetting valve system was fabricated using low-cost materials and techniques, can be actuated by a battery, and can be integrated into portable microfluidic devices suitable for point-of-care analysis in resource-limited settings.     

Nanomechanical Protein Concentration Detector Using a Nanogap Squeezing Actuator With Compensated Displacement Monitoring Electrodes

We present a new class of the protein concentration detector based on the mechanical stiffness measurement of protein-receptor layers in a squeezed nanogap. Compared to the previous protein size detector, the present device reduces the distortion and uncertainty in the displacement measurement by adding an actuated nanogap and reference electrodes. Compared to the conventional protein detectors based on electrochemical, optical, optomechanical, and mechanical principles, the present device also offers simple, inexpensive, and high-precision protein detection. We design and fabricate the protein concentration detector using an electrothermal actuator and two nanogaps with reference electrodes. In an experimental study, we verify that the present protein detector measures the size of the proteins streptavidin and m-antibiotin as 12.1 plusmn 2.3 and 13.2 plusmn 3.3 nm at the measurement uncertainty of plusmn1.9 nm, respectively, while showing the concentration detection sensitivity of 2.88 N/m/nM in the m-antibiotin concentration range of 5-10 nM.     

协同DNA计算机解空间问题的模块化设计方案

提出了一种基于检测型生物芯片的协同DNA计算机解空间问题的模块化解决方案。为了解决解空间检测这个问题，该文总结了目前典型的DNA计算模型中所用到的生物检测技术，在先前的协同DNA计算机基本组成原理模型的基础上，结合了当前检测型生物芯片技术的发展趋势，提出了解决方案，并对各模块的工作原理、功能等进行了介绍，给出了问题与展望。     

Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide

Development of an electrochemical biosensor based on peptide nucleic acid (PNA) probe for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene corresponding oligonucleotide using methylene blue (MB) as an electrochemical indicator is described. The interaction between MB and short sequence of p53, one of the most important tumor suppressor genes due to its dysfunction in the majority of human cancers, was studied by differential pulse voltammety (DPV). Probe modified electrode was prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of gold electrode (GE). The hybridization of PNA probe with target DNA was performed in solution to form PNA-DNA hybrid on the surface of the GE. A significant increase in the reduction signal of MB was observed upon hybridization of the probe with the complementary DNA. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Furthermore, our results confirmed the ability of the sensor to detect single base mismatch in the sample oligonucleotide. The influence of probe concentration on the effective discrimination against noncomplementary sequence and point mutation was also investigated. Diagnostic performance of the biosensor is described and the detection limit is found 6.82 × 10⁻¹⁰ M. The electrochemical impedance spectroscopy was also employed to further investigate the sensor function.     

Electrical detection of DNA hybridization with multilayer gold nanoparticles between nanogap electrodes

This study presents a DNA detection method via hybridization of a probe oligonucleotide with a target DNA and with a substrate oligonucleotide, which leads to self-assembly of gold nanoparticles and a change in the observed current. In contrast to previously reported methods, the present method can be used to detect a target DNA without a silver enhancement step. The device can be washed with 0.3 M PBS below 50 °C for a few minutes, indicating the stringency of target DNA hybridization. Target DNA concentration of as low as 10 picomolar can be detected by this method. Importantly, identification of single-base-pair mismatch in the target DNA can be accomplished with this nano-gap DNA chip.The authors thank for Shu-Fen, Hos group and her assistant, Mr. Wong for useful discussions. Dr. Ho has also been supported in measuring the electricity properties.