Thomas Ming Hung Lee

A DNA biochip for on-the-spot multiplexed pathogen identification

Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole−probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

An improved anodic bonding process using pulsed voltage technique

In this study, we report a pulsed-voltage technique that is commonly employed in the electroplating industry to achieve a more efficient Si-glass anodic bonding process than the conventional constant electric field process. This technique features a less stringent voltage requirement and a shortened bonding time without compromising the tensile strength of the bonded structure. A square waveform voltage profile is used to investigate the effects of pulsed-voltage profile on the bonding time. In particular, the effects of magnitude of the base voltage and duration of the peak and base voltages are investigated. With peak and base voltages set to 400 and 300 V, respectively, and the duration of each voltage pulse fixed at 10-30 s, the bonding time is reduced to 30% of that required by a constant field process (400 V). Tensile strength of all completely bonded Si-glass pairs prepared by this technique is greater than 15 MPa. A postulated bonding mechanism based on the experimental results is presented.

Over-the-Counter Biosensors: Past, Present, and Future.

The demand for specific, low cost, rapid, sensitive and easy detection of biomolecules is huge. A well-known example is the glucose meters used by diabetics to monitor their blood glucose levels. Nowadays, a vast majority of the glucose meters are based on electrochemical biosensor technology. The inherent small size and simple construction of the electrochemical transducer and instrument are ideally suited for point-of-care biosensing. Besides glucose, a wide variety of electrochemical biosensors have been developed for the measurements of some other key metabolites, proteins, and nucleic acids. Nevertheless, unlike the glucose meters, limited success has been achieved for the commercialization of the protein and nucleic acid biosensors. In this review article, key technologies on the electrochemical detection of key metabolites, proteins, and DNAs are discussed in detail, with particular emphasis on those that are compatible to home-use setting. Moreover, emerging technologies of lab-on-a-chip microdevices and nanosensors (i.e., silicon and carbon nanotube field-effect sensors) offer opportunities for the construction of new generation biosensors with much better performances. Together with the continuous innovations in the basic components of biosensors (i.e., transducers, biorecognition molecules, immobilization and signal transduction schemes), consumers could soon buy different kinds of biosensing devices in the pharmacy stores.

Detailed characterization of anodic bonding process between glass and thin-film coated silicon substrates

Abstract Anodic bonding between Si-based and glass substrates has been characterized in detail. The effects of magnitude of the applied voltage, surface properties (coating of Si substrate), and surface cleanliness (pre-bonding cleaning procedure) on the time required for complete bonding were thoroughly studied. First, the generic bonding time versus applied voltage plot was found to be concave in shape (viewed from the origin). For bonding between p-type Si substrate and Corning 7740 glass pre-cleaned with acetone, the time required was cut down from 38 to 4 min if the applied voltage was increased from 200 to 500 V. Second, the bonding time required for five Si-based substrates in ascending order was determined to be Si (p-type), polysilicon, silicon nitride, silicon oxide and then Si (n-type). Third, the bonding between p-type Si substrate, pre-cleaned with H2SO4–H2O2 and HF, and Corning 7740 glass was completed within 1 min, which was much faster than that pre-cleaned with acetone (4 min). Finally, from bonding point of view, Corning 7740 glass was superior to Corning 7059 glass and Fisher slide due to its thermal coefficient of expansion matching with the underlying Si substrate and the presence of significant amount of sodium ions in the glass.

Immobilization-free sequence-specific electrochemical detection of DNA using ferrocene-labeled peptide nucleic acid.

An electrochemical method for sequence-specific detection of DNA without solid-phase probe immobilization is reported. This detection scheme starts with a solution-phase hybridization of ferrocene-labeled peptide nucleic acid (Fc-PNA) and its complementary DNA (cDNA) sequence, followed by the electrochemical transduction of Fc-PNA-DNA hybrid on indium tin oxide (ITO)-based substrates. On the bare ITO electrode, the negatively charged Fc-PNA-DNA hybrid exhibits a much reduced electrochemical signal than that of the neutral-charge Fc-PNA. This is attributed to the electrostatic repulsion between the negatively charged ITO surface and the negatively charged DNA, hindering the access of Fc-PNA-DNA to the electrode. On the contrary, when the transduction measurement is done on the ITO electrode coated with a positively charged poly(allylamine hydrochloride) (PAH) layer, the electrostatic attraction between the (+) PAH surface and the (-) Fc-PNA-DNA hybrid leads to a much higher electrochemical signal than that of the Fc-PNA. The measured electrochemical signal is proportional to the amount of cDNA present. In terms of detection sensitivity, the PAH-modified ITO platform was found to be more sensitive (with a detection limit of 40 fmol) than the bare ITO counterpart (with a detection limit of 500 fmol). At elevated temperatures, this method was able to distinguish fully matched target DNA from DNA with partial mismatches. Unpurified PCR amplicons were detected using a similar format with a detection limit down to 4.17 amol. This detection method holds great promise for single-base mismatch detection as well as electrochemistry-based detection of post-PCR products.

DNA-based bioanalytical microsystems for handheld device applications

Abstract This article reviews and highlights the current development of DNA-based bioanalytical microsystems for point-of-care diagnostics and on-site monitoring of food and water. Recent progresses in the miniaturization of various biological processing steps for the sample preparation, DNA amplification (polymerase chain reaction), and product detection are delineated in detail. Product detection approaches utilizing “portable” detection signals and electrochemistry-based methods are emphasized in this work. The strategies and challenges for the integration of individual processing module on the same chip are discussed.

Precise temperature control of microfluidic chamber for gas and liquid phase reactions

A silicon-based micromachined fluidic chamber with integrated platinum heaters and sensors has been developed and thermally well characterized. This device is highly applicable to both gas and liquid phase reactions that require excellent thermal control. The chamber is thermally isolated from the bulk silicon by a thin silicon nitride membrane resulting in low power consumption. The digitally feedback controlled device demonstrates the ability of precise temperature control, excellent temperature uniformity, rapid heating and cooling. These thermal characteristics are ascribable to the success of miniaturized reaction chamber and Micro Total Analysis Systems (μTAS). In addition, gain scheduling control algorithm in conjunction with normal feedback proportional and integral (PI) scheme was implemented to provide better thermal cycling performance. 3D numerical simulation was also conducted to map the spatial temperature distribution within the miniaturized fluidic device. Simulation results and experimental data show good agreements.

Effects of gold nanoparticle and electrode surface properties on electrocatalytic silver deposition for electrochemical DNA hybridization detection

In this paper we report the catalytic effects of various gold nanoparticles for silver electrodeposition on indium tin oxide (ITO)-based electrodes, and successfully apply this methodology for signal amplification of the hybridization assay. The most widely used gold nanoparticle-based hybridization indicators all promote silver electrodeposition on the bare ITO electrodes, with decreasing catalytic capability in order of 10 nm gold, DNA probe-10 nm gold conjugate, streptavidin-5 nm gold, and streptavidin-10 nm gold. Of greater importance, these electrocatalytic characteristics are affected by any surface modifications of the electrode surfaces. This is illustrated by coating the ITO with an electroconducting polymer, poly(2-aminobenzoic acid)(PABA), as well as avidin molecules, which are promising immobilization platforms for DNA biosensors. The catalytic silver electrodeposition of the gold nanoparticles on the PABA-coated ITO surfaces resembles that on the bare surfaces. With avidin covalently bound to the PABA, it is interesting to note that the changes in electrocatalytic performance vary for different types of gold nanoparticles. For the streptavidin-5 nm gold, the silver electrodeposition profile is unaffected by the presence of the avidin layer, whereas for both the 10 nm Au and DNA probe-10 nm gold conjugate, the deposition profiles are suppressed. The streptavidin-5 nm gold is employed as the hybridization indicator, with avidin-modified (via PABA) ITO electrode as the immobilization platform, to enable signal amplification by the silver electrodeposition process. Under the conditions, this detection strategy offers a signal-to-noise ratio of 20. We believe that this protocol has great potential for simple, reproducible, highly selective and sensitive DNA detection on fully integrated microdevices in clinical diagnostics and environmental monitoring applications.

Sharp tipped plastic hollow microneedle array by microinjection moulding

A method of producing sharp tipped plastic hollow microneedle arrays using microinjection moulding is presented in this paper. Unlike traditional approaches, three mould inserts were used to create the sharp tips of the microneedles. Mould inserts with low surface roughness were fabricated using a picosecond laser machine. Sharp tipped plastic hollow microneedles 500 µm in height were fabricated using a microinjection moulding machine developed by the authors’ group. In addition, the strength of the microneedle was studied by simulation and penetration experiments. Results show that the microneedles can penetrate into skin, delivering liquid successfully without any breakage or severe deformation. Techniques presented in this paper can be used to fabricate sharp tipped plastic hollow microneedle arrays massively with low cost.