Pin Chuan Chen

Fully Integrated Thermoplastic Genosensor for the Highly Sensitive Detection and Identification of Multi‐Drug‐Resistant Tuberculosis

Infectious diseases are a major global health burden accounting for approximately 15 million deaths annually, many from drug resistant pathogenic agents, with a significant number of cases occurring in developing countries.[1–7] In particular, the resurgence of tuberculosis (TB) has been accompanied by the rapid spread of multi-drug resistance TB (MDR-TB) resulting from Mycobacterium tuberculosis (Mtb) strains that fail to respond to the first-line drugs, rifampin and isoniazid. Currently, <5% of ~0.5 million MDR-TB cases estimated globally are appropriately diagnosed and treated due in part to the long assay turnaround time associated with conventional culture-based drug susceptibility testing.[8]

Titer plate formatted continuous flow thermal reactors for high throughput applications: fabrication and testing

A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 × 8 mm2, matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a λ-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as ~5 min at a flow velocity of 3 mm s−1 (15.3 s cycle−1) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s−1 (46.2 s cycle−1) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

Passive alignment structures in modular, polymer microfluidic devices

For connecting polymeric, modular microfluidic devices, precise, passive alignment structures can prevent infinitesimal motions between the devices and minimize misalignment of the devices. The motion and constraint of passive alignment structures were analyzed for the design of assembly features using screw theory. A combination of three v-groove and sphere joints constrained all degrees of freedom of the two mating plates without over-constraint. To validate the designed passive alignment scheme, hot embossing experiments were conducted using a micromilled brass mold insert, containing alignment features. Prototype alignment structures have dimensional variation. The alignment accuracy of the stacked polymeric plates was estimated by the mismatches between alignment marks of two plates. The mismatches ranged from 28 μm to 70 μm.Copyright

Replication of Reliable Assembly Features for Polymer Modular Microfluidic Systems

BioMEMS are compact devices which use microfabrication to miniaturize conventional benchtop instruments. The benefits of using micro devices are the need for less chemical reagents, faster processing, and portability. Realizing a powerful sample-to-answer micro system requires more than just microfabrication technology; thermal management, microfluidic control, and interfacing technology must also be considered. You et al. [1] applied kinematic constraint analysis using screw theory to design the assembly features for passive alignment structures for polymer, modular microfluidic devices. Three pairs of v-groove and hemispherical pin joints were chosen as kinematic pairs for mating two modules. Dimensional variation of the assembly features was one of principal contributions to the mismatch of 28 μm-75 μm between stacked plates in assembly that was reported. This was primarily a result of incomplete mold filling and deformation of the passive alignment structures during the demolding process. Hemisphere-tipped recesses, with additional annular walls as dummy structures, were used to designed to achieve better replication by improving polymer filling and reducing the deformation during demolding. In prototype devices, the posts with the annular dummy structures had a mean height of 922.2 μm – 924.1 μm while the original posts without dummy structures had mean heights of 865.3 μm – 891.2 μm in 10 samples under the same embossing conditions for a design height of 925 μm. Alignment accuracy of better than 10 μm was achieved in the assembly of two plates.Copyright

Protein Adsorption in a Continuous Flow Microchannel Environment

Protein adsorption is a critical issue in microfluidic devices especially for those reactions depending on proteins like the polymerase chain reaction (PCR). Understanding protein absorption phenomena in different geometry microchannels and evaluating the efficiency of dynamic coating, which has been using as a method to prevent protein adsorption, are important tasks. Two different sets of microchannels were designed and fabricated on polymers. Bovine serum albumin (BSA) was used as a model protein for quantification of and monitoring the protein loss in different microchannel geometries. Up to 58% of the BSA was lost after flowing a 2030 mm long microchannel. The BSA adsorption rate changed along the microchannel. Smaller microchannels required a longer time to achieve protein saturation point. Dynamic coating was shown to be a time consuming and inefficient method to prevent protein adsorption in a continuous flow environment.Copyright

Assessment and improvement of the thermal performance of a polycarbonate micro Continuous Flow Polymerase Chain Reactor (CFPCR)

BioMEMS are compact devices that use microfabrication to miniaturize benchtop instrumentation. Due to the requirement for uniform temperature distributions over restricted areas, thermal isolation, and faster heating and cooling rates in a limited space, thermal management is a key to ensuring successful performance of BioMEMS devices. The continuous flow polymerase chain reactor (CFPCR) is a compact BioMEMS device that is used to amplify target DNA fragments using repeated thermal cycling. The temperature distribution on the backside of a micro CFPCR was measured using thermochromic liquid crystals and an infrared camera. In the liquid crystal experiment, the performance of a 5 mm thick polycarbonate micro CFPCR with thin film heaters attached directly to the bottom polycarbonate surface over each temperature zone was studied. Natural convection was used as a cooling mechanism. The temperature distribution in the renaturation zone was dependent on the positions of the feedback thermocouples in each zone. Three different thermocouple configurations were assessed and the liquid crystal images showed that a best case 3.86°C temperature difference across the zone, leading to a 20% amplification efficiency compared to a commercial thermal cycler [5]. The device was modified to improve the temperature distribution: a thinner substrate, 2 mm, reduced the thermal capacitance; grooves were micro-milled in the backside to isolate each temperature zone; and three separate copper heating stages, combining the thin film heaters with copper plates, applied uniform temperatures to each zone [10]. Infrared camera images showed that the temperature distributions were distinct and uniform with a ±0.3 °C variations in each temperature zone, improving amplification efficiency to 72%. Good thermal management for PCR amplification can’t only increase its reliability and yield efficiency, but also accelerate the entire analytical process.Copyright

Performance of an Electrokinetic Shuttle Polymerase Chain Reactor

An alternative polymerase chain reactor (PCR) driven by electrokinetic flow was developed and tested. A single straight microchannel and a double-T intersection were designed for injection of DNA samples and thermal cycling by shuttling between constant temperature zones. Thermal performance of the device was studied using numerical and analytical models to understand the temperature distribution. Devices were made on a polycarbonate substrate by hot embossing with a micromilled brass mold insert. A PID control system, with a tolerance of ± 0.2°C, was used to maintain the temperatures in each zone during experiments. Power consumption in each zone was predicted using thermal simulations. Molecular diffusion of 500 bp DNA was evaluated using two methods, an empirical equation and an analytical model, and the diffusion length after 20 cycles from both models was 100 μm with a 0.97 μm difference. Electroosmotic flow (EOF) was minimized by using dynamic coating and Joule heating was reduced by decreasing the KCl component in the DNA cocktail. Successful amplification of 500 bp DNA fragments at shuttle velocities of 1mm s-1 (620 seconds), 2mm s-1 (310 seconds), and 3mm s-1 (207 seconds) was demonstrated for 20 thermal cycles. The amplification efficiencies were 31%, 28%, and 18%, respectively. Unintentional flows resulting from siphoning phenomenon due to hydrostatic pressure, and Laplace pressure due to surface tension, may be responsible for the reduced amplification performance.Copyright