Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection.

BACKGROUND: High-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. RESULT: Automatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 µL droplet on MALDI plate, high recovery (76-116%) and rapid evaporation in 2 min. The analytes were concentrated 4-8 times, forming even crystallization, reaching the detection limit at the concentration of 50 µg L-1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%-114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SIGNIFICANCE: The ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+-Translocating Pyrophosphatases Through Hydrogen-Deuterium Exchange Mass Spectrometry.

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

Spontaneous vortex formation by microswimmers with retarded attractions.

Collective states of inanimate particles self-assemble through physical interactions and thermal motion. Despite some phenomenological resemblance, including signatures of criticality, the autonomous dynamics that binds motile agents into flocks, herds, or swarms allows for much richer behavior. Low-dimensional models have hinted at the crucial role played in this respect by perceived information, decision-making, and feedback, implying that the corresponding interactions are inevitably retarded. Here we present experiments on spherical Brownian microswimmers with delayed self-propulsion toward a spatially fixed target. We observe a spontaneous symmetry breaking to a transiently chiral dynamical state and concomitant critical behavior that do not rely on many-particle cooperativity. By comparison with the stochastic delay differential equation of motion of a single swimmer, we pinpoint the delay-induced effective synchronization of the swimmers with their own past as the key mechanism. Increasing numbers of swimmers self-organize into layers with pro- and retrograde orbital motion, synchronized and stabilized by steric, phoretic, and hydrodynamic interactions. Our results demonstrate how even most simple retarded interactions can foster emergent complex adaptive behavior in small active-particle ensembles.

Slab waveguide-based particle plasmon resonance optofluidic biosensor for rapid and label-free detection.

An effective bio-sensing platform that would meet the criteria of rapid, simple, and sensitive detection is crucial to translate bench research to clinical applications. However, simultaneously rapid and sensitive biosensing remains challenging for practical biomedical applications. In this study, for the first time, we demonstrate a cost-effective, label-free, real-time, and sensitive slab waveguide-based particle plasmon resonance (WGPPR) biosensor for practical clinical applications. A suspended glass slab waveguide structure with excellent optical confinement properties was designed and fabricated as the biosensor. Gold nanoparticles (AuNPs) were deposited on the top surface of the waveguide layer to significantly enhance the optical near field through the localized surface plasmon resonance (LSPR) effect. When light travels through the waveguide, the change in the local refractive index (RI) near the surface of the AuNPs can be transformed into changes in the intensity of transmitted light, thereby enabling sensitive and real-time detection. The RI sensing experiment shows a good sensor resolution of 1.43 × 10-4 RIU, which represents a 395% enhancement compared to that of the sensor without AuNPs. Through biochemical detection experiments, we measured IgG and determined the detection limit (LOD) at 614 ng mL-1 in ∼4 min, thereby proving the feasibility of the bio-detection sensing functionality. This study demonstrates a new type of WGPPR biosensor, which offers several unique advantages such as simple structure, high sensitivity, and rapid bio-sensing for practical bio-medical sensing applications. The new biosensor also fulfils point-of-care (POC) requirements.

Advanced Manufacturing in the Fabrication of a Lifelike Brain Glioblastoma Simulator for the Training of Neurosurgeons.

Neurosurgeons require considerable expertise and practical experience to deal with the critical situations commonly encountered in complex surgical operations such as cerebral cancer; however, trainees in neurosurgery seldom have the opportunity to develop these skills in the operating room. Physical simulators can give trainees the experience they require. In this study, we adopted advanced molding and replication techniques in the fabrication of a physical simulator for use in practicing the removal of cerebral tumors. Our combination of additive manufacturing and molding technology with elastic material casting made it possible to create a simulator that realistically mimics the skull, brain stem, soft brain lobes, and cerebral cancer with cerebral tumors located precisely where they are likely to appear. Multiple and systematic experiments were conducted to prove that the elastic material used herein was appropriated for building professional medical physical simulator. One neurosurgical trainee reported that under the guidance of a senior neurosurgeon, the physical simulator helped to elucidate the overall process of cerebral cancer removal and provided a realistic impression of the tactile feelings involved in craniotomy. The trainee also learned how to make decisions when facing the infiltration of a cerebral tumor into normal brain lobes. Our results demonstrate the efficacy of the proposed physical simulator in preparing trainees for the rigors involved in performing highly delicate surgical operations.

Using Stereolithographic Printing to Manufacture Monolithic Microfluidic Devices with an Extremely High Aspect Ratio.

Stereolithographic printing (SL) is widely used to create mini/microfluidic devices; however, the formation of microchannels smaller than 500 µm with good inner surface quality is still challenging due to the printing resolution of current commercial printers and the z-overcure error and scalloping phenomena. In the current study, we used SL printing to create microchannels with the aim of achieving a high degree of dimensional precision and a high-quality microchannel inner surface. Extensive experiments were performed and our results revealed the following: (1) the SL printing of microchannels can be implemented in three steps including channel layer printing, an oxygen inhibition process, and roof layer printing; (2) printing thickness should be reduced to minimize the scalloping phenomenon, which significantly improves dimensional accuracy and the quality of inner microchannel surfaces; (3) the inclusion of an oxygen inhibition step is a critical and efficient approach to suppressing the z-overcure error in order to eliminate the formation of in-channel obstructions; (4) microchannels with an extremely high aspect ratio of 40:1 (4000 µm in height and 100 µm in width) can be successfully manufactured within one hour by following the three-step printing process.

Leakage pressures for gasketless superhydrophobic fluid interconnects for modular lab-on-a-chip systems.

Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of liquids between component chips and to/from microfluidic systems. Unfortunately, most interconnect designs add additional physical constraints to chips with each additional interconnect leading to over-constrained microfluidic systems. The competing constraints provided by multiple interconnects induce strain in the chips, creating indeterminate dead volumes and misalignment between chips that comprise the microfluidic system. A novel, gasketless superhydrophobic fluidic interconnect (GSFI) that uses capillary forces to form a liquid bridge suspended between concentric through-holes and acting as a fluid passage was investigated. The GSFI decouples the alignment between component chips from the interconnect function and the attachment of the meniscus of the liquid bridge to the edges of the holes produces negligible dead volume. This passive seal was created by patterning parallel superhydrophobic surfaces (water contact angle ≥ 150°) around concentric microfluidic ports separated by a gap. The relative position of the two polymer chips was determined by passive kinematic constraints, three spherical ball bearings seated in v-grooves. A leakage pressure model derived from the Young-Laplace equation was used to estimate the leakage pressure at failure for the liquid bridge. Injection-molded, Cyclic Olefin Copolymer (COC) chip assemblies with assembly gaps from 3 to 240 µm were used to experimentally validate the model. The maximum leakage pressure measured for the GSFI was 21.4 kPa (3.1 psig), which corresponded to a measured mean assembly gap of 3 µm, and decreased to 0.5 kPa (0.073 psig) at a mean assembly gap of 240 µm. The effect of radial misalignment on the efficacy of the gasketless seals was tested and no significant effect was observed. This may be a function of how the liquid bridges are formed during the priming of the chip, but additional research is required to test that hypothesis.

Development of an Automated Optical Inspection System for Rapidly and Precisely Measuring Dimensions of Embedded Microchannel Structures in Transparent Bonded Chips.

This study aimed to develop an automated optical inspection (AOI) system that can rapidly and precisely measure the dimensions of microchannels embedded inside a transparent polymeric substrate, and can eventually be used on the production line of a factory. The AOI system is constructed based on Snell's law. The concept holds that, when light travels through two transparent media (air and the microfluidic chip transparent material), by capturing the parallel refracted light from a light source that went through the microchannel using a camera with a telecentric lens, the image can be analyzed using formulas derived from Snell's law to measure the dimensions of the microchannel cross-section. Through the NI LabVIEW 2018 SP1 programming interface, we programmed this system to automatically analyze the captured image and acquire all the needed data. The system then processes these data using custom-developed formulas to calculate the height and width measurements of the microchannel cross-sections and presents the results on the human-machine interface (HMI). In this study, a single and straight microchannel with a cross-sectional area of 300 µm × 300 µm and length of 44 mm was micromachined and sealed with another polymeric substrate by a solvent bonding method for experimentations. With this system, 45 cross-sectional areas along the straight microchannel were measured within 20 s, and experiment results showed that the average measured error was less than 2%.

Engineering Additive Manufacturing and Molding Techniques to Create Lifelike Willis' Circle Simulators with Aneurysms for Training Neurosurgeons.

Neurosurgeons require considerable expertise and practical experience in dealing with the critical situations commonly encountered during difficult surgeries; however, neurosurgical trainees seldom have the opportunity to develop these skills in the operating room. Therefore, physical simulators are used to give trainees the experience they require. In this study, we created a physical simulator to assist in training neurosurgeons in aneurysm clipping and the handling of emergency situations during surgery. Our combination of additive manufacturing with molding technology, elastic material casting, and ultrasonication-assisted dissolution made it possible to create a simulator that realistically mimics the brain stem, soft brain lobes, cerebral arteries, and a hollow transparent Circle of Willis, in which the thickness of vascular walls can be controlled and aneurysms can be fabricated in locations where they are likely to appear. The proposed fabrication process also made it possible to limit the error in overall vascular wall thickness to just 2-5%, while achieving a Young's Modulus closely matching the characteristics of blood vessels (~5%). One neurosurgical trainee reported that the physical simulator helped to elucidate the overall process of aneurysm clipping and provided a realistic impression of the tactile feelings involved in this delicate operation. The trainee also experienced shock and dismay at the appearance of leakage, which could not immediately be arrested using the clip. Overall, these results demonstrate the efficacy of the proposed physical simulator in preparing trainees for the rigors involved in performing highly delicate neurological surgical operations.

Tunable microlens array fabricated by a silicone oil-induced swelled polydimethylsiloxane (PDMS) membrane bonded to a micro-milled microfluidic chip.

Microlens arrays (MLAs) nowadays are critical micro-optical components and they can be applied in many application fields, such as optical communication systems and flat panel display modules. This article describes a novel approach to the fabrication of tunable, highly reliable, and uniform polydimethylsiloxane (PDMS) MLAs. A polydimethylsiloxane (PDMS) membrane is bonded to a micro-milled poly(methyl methacrylate) (PMMA) microfluidic chip and exposed to silicone oil of a specific viscosity. Molecules in the oil insert themselves into the molecular structure of the PDMS membrane, causing it to swell and subsequently form dome-shaped MLAs. From our experiments, we derived the following conclusions. First, the homogeneous swelling of the PDMS resulted in MLAs with a high numerical aperture (0.5), high uniformity illumination (CV of the illumination intensity is between 2.5%∼5.1%), and high uniformity (CV of sag height of MLAs is less than 0.05). Second, the shorter molecular chains in low-viscosity oils diffused more readily into the PDMS membrane, which increased the effects on swelling, resulting in MLAs with higher sag height and higher numerical aperture. For example, the 5 cst silicone oil resulted in sag height of 191 µm with NA of 0.50, whereas the 100 cst silicone oil resulted in sag height of 86 µm with numerical aperture of 0.33. Finally, the integrated mixer module enabled the simultaneous tuning of the 7 × 7 MLAs simply by adjusting the injection flow rates of the constituent silicone oils.

Using Micromachined Molds, Partial-curing PDMS Bonding Technique, and Multiple Casting to Create Hybrid Microfluidic Chip for Microlens Array.

In a previous study, we presented a novel manufacturing process for the creation of 6 × 6 and 8 × 8 microlens arrays (MLAs) comprising lenses with diameters of 1000 µm, 500 µm, and 200 µm within an area that covers 10 mm × 10 mm. In the current study, we revised the manufacturing process to allow for the fabrication of MLAs of far higher density (15 × 15 and 29 × 29 within the same area). In this paper, we detail the revised manufacturing scheme, including the micromachining of molds, the partial-curing polydimethylsiloxane (PDMS) bonding used to fuse the glass substrate and PDMS, and the multi-step casting process. The primary challenges that are involved in creating MLAs of this density were ensuring uniform membrane thickness and preventing leakage between the PDMS and glass substrate. The experiment results demonstrated that the revised fabrication process is capable of producing high density arrays: Design I produced 15 × 15 MLAs with lens diameter of 0.5 mm and fill factor of 47.94%, while Design II produced 29 × 29 MLAs with lens diameter of 0.25 mm and fill factor of 40.87%. The partial-curing PDMS bonding system also proved to be effective in fusing PDMS with glass (maximum bonding strength of approximately six bars). Finally, the redesigned mold was used to create PDMS membranes of high thickness uniformity (coefficient of variance <0.07) and microlenses of high lens height uniformity (coefficient of variance <0.15).

Simple and low-cost production of hybrid 3D-printed microfluidic devices.

The use of three-dimensional (3D) printing for the fabrication of microfluidic chips has attracted considerable attention among researchers. This low-cost fabrication method allows for rapid prototyping and the creation of complex structures; however, these devices lack optical transparency, which greatly hinders the characterization and quantification of experiment results. To address this problem, integrating a transparent substrate with a 3D-printed chip is an effective approach. In this study, we present a solvent bonding method of poly(methyl methacrylate) (PMMA) and acrylonitrile butadiene styrene (ABS) thermoplastic materials for the creation of optically detectable 3D-printed microfluidic devices. To achieve an excellent bonding between PMMA and ABS substrates, we used spray coating as a method for the distribution of ethanol solution followed by UV exposure and post-annealing step to improve the bonding strength. We fabricated a microfluidic chip with S-microchannel to characterize the bonding protocol, and other two application-oriented microfluidic chips, including a 3D split-and-recombine-based passive micromixer, and an integrated microchip for the mixing of two streams of liquid prior to the formation of double-emulsion droplets, to evaluate the efficacy of the proposed scheme. As a result, at least eight bars of the bonding strength between PMMA/ABS substrates was achieved, and the ability of producing optically detectable 3D-printed microfluidic devices based on this bonding method was confirmed.

Microfabrication of Nonplanar Polymeric Microfluidics.

For four decades, microfluidics technology has been used in exciting, state-of-the-art applications. This paper reports on a novel fabrication approach in which micromachining is used to create nonplanar, three-dimensional microfluidic chips for experiments. Several parameters of micromachining were examined to enhance the smoothness and definition of surface contours in the nonplanar poly(methyl methacrylate) (PMMA) mold inserts. A nonplanar PMMA/PMMA chip and a nonplanar polydimethylsiloxane (PDMS)/PMMA chip were fabricated to demonstrate the efficacy of the proposed approach. In the first case, a S-shape microchannel was fabricated on the nonplanar PMMA substrate and sealed with another nonplanar PMMA via solvent bonding. In the second case, a PDMS membrane was casted from two nonplanar PMMA substrates and bonded on hemispherical PMMA substrate via solvent bonding for use as a microlens array (MLAs). These examples demonstrate the effectiveness of micromachining in the fabrication of nonplanar microfluidic chips directly on a polymeric substrate, as well as in the manufacture of nonplanar mold inserts for use in creating PDMS/PMMA microfluidic chips. This technique facilitates the creation of nonplanar microfluidic chips for applications requiring a three-dimensional space for in vitro characterization.

Optofluidic refractive-index sensors employing bent waveguide structures for low-cost, rapid chemical and biomedical sensing.

We propose and develop an intensity-detection-based refractive-index (RI) sensor for low-cost, rapid RI sensing. The sensor is composed of a polymer bent ridge waveguide (BRWG) structure on a low-cost glass substrate and is integrated with a microfluidic channel. Different-RI solutions flowing through the BRWG sensing region induce output optical power variations caused by optical bend losses, enabling simple and real-time RI detection. Additionally, the sensors are fabricated using rapid and cost-effective vacuum-less processes, attaining the low cost and high throughput required for mass production. A good RI solution of 5.31 10-4 × RIU-1 is achieved from the RI experiments. This study demonstrates mass-producible and compact RI sensors for rapid and sensitive chemical analysis and biomedical sensing.

Microfabricated microfluidic platforms for creating microlens array.

The paper presents a novel and economic manufacturing process for microlens arrays (MLAs). This process uses micromilling machining, PDMS casting, and hybrid bonding between a glass substrate and PDMS membrane to create a microfluidic chip which is used for manufacturing MLAs on a PDMS substrates. MLAs of various diameters were fabricated for experiments, including 1000 µm, 500 µm, and 200 µm. The sag height of the MLAs is easily adjusted by controlling the pressure inside the microchannel to deform the PDMS membrane. Multiple experiments were conducted to characterize the performance of MLAs, the results of which demonstrate: (1) this fabrication process is able to manufacture MLAs with various dimensions and the diameter of an MLAs is determined by the size of micromilling bit and cutting path; (2) the sag height and curvature of MLAs can be controlled by the PDMS membrane thickness and the hydraulic pressure inside the microchannel; (3) an optical system was built to investigate the uniformity of MLAs and the experiment results showed uniform focal length of MLAs; (4) the resulting MLAs magnify tiny objects and significantly enhance the fluorescence signal emitted from the microchannel.

Micromachining Microchannels on Cyclic Olefin Copolymer (COC) Substrates with the Taguchi Method.

Micromilling is a straightforward approach to the manufacture of polymer microfluidic devices for applications in chemistry and biology. This fabrication process reduces costs, provides a relatively simple user interface, and enables the fabrication of complex structures, which makes it ideal for the development of prototypes. In this study, we investigated the influence of micromilling parameters on the surface roughness of a cyclic olefin copolymer (COC) substrate. We then employed factor analysis to determine the optimal cutting conditions. The parameters used in all experiments were the spindle speed, the feed rate, and the depth of cut. Roughness was measured using a stylus profilometer. The lowest roughness was 0.173 µm at a spindle speed of 20,000 rpm, feed rate of 300 mm/min, and cut depth of 20 µm. Factor analysis revealed that the feed rate has the greatest impact on surface quality, whereas the depth of cut has the least impact.

Characterization of thermoplastic microfiltration chip for the separation of blood plasma from human blood.

In this study, we developed a fully thermoplastic microfiltration chip for the separation of blood plasma from human blood. Spiral microchannels were manufactured on a PMMA substrate using a micromilling machine, and a commercial polycarbonate membrane was bonded between two thermoplastic substrates. To achieve an excellent bonding between the commercial membrane and the thermoplastic substrates, we used a two-step injection and curing procedure of UV adhesive into a ring-shaped structure around the microchannel to efficiently prevent leakage during blood filtration. We performed multiple filtration experiments using human blood to compare the influence of three factors on separation efficiency: hematocrit level (40%, 23.2%, and 10.9%), membrane pore size (5 µm, 2 µm, and 1 µm), and flow rate (0.02 ml/min, 0.06 ml/min, 0.1 ml/min). To prevent hemolysis, the pressure within the microchannel was kept below 0.5 bars throughout all filtration experiments. The experimental results clearly demonstrated the following: (1) The proposed microfiltration chip is able to separate white blood cells and red blood cells from whole human blood with a separation efficiency that exceeds 95%; (2) no leakage occurred during any of the experiments, thereby demonstrating the effectiveness of bonding a commercial membrane with a thermoplastic substrate using UV adhesive in a ring-shaped structure; (3) separation efficiency can be increased by using a membrane with smaller pore size, by using diluted blood with lower hematocrit, or by injecting blood into the microfiltration chip at a lower flow rate.

Effect of surfactant and budesonide on the pulmonary distribution of fluorescent dye in mice.

BACKGROUND: Surfactant is a useful vehicle for the intratracheal delivery of medicine to the distal lung. The aim of this study was to analyze the effect of intratracheal surfactant and budesonide instillation on the pulmonary distribution of fluorescent dye in mice. METHODS: Male athymic nude mice were assigned randomly as controls, fluorescent dye, fluorescent dye + surfactant (50 mg/kg), fluorescent dye + budesonide (0.25 mg/kg), and fluorescent dye + surfactant + budesonide groups. A total volume of 60 µL fluorescent solutions was intratracheally injected and followed by 60 µL of air. We photographed and measured fluorescence in the lungs, from the back, 15 minutes after intratracheal administration using an IVIS Xenogen imaging instrument. RESULTS: The fluorescent dye (1,1'-dioctadecyltetramethyl indotricarbocyanine iodide) was most strongly detected near the trachea and weakly detected in the lungs in mice administered with fluorescent solutions. Almost no fluorescence was seen in the lung region of control mice. Intratracheal administration of surfactant or budesonide increased fluorescent intensity compared with control mice. Combined administration of surfactant and budesonide further increased fluorescent intensity compared with mice given surfactant or budesonide alone. CONCLUSION: Surfactant and budesonide enhance the pulmonary distribution of fluorescent dye in mice.

Titer-plate formatted continuous flow thermal reactors: Design and performance of a nanoliter reactor.

Arrays of continuous flow thermal reactors were designed, configured, and fabricated in a 96-device (12 × 8) titer-plate format with overall dimensions of 120 mm × 96 mm, with each reactor confined to a 8 mm × 8 mm footprint. To demonstrate the potential, individual 20-cycle (740 nL) and 25-cycle (990 nL) reactors were used to perform the continuous flow polymerase chain reaction (CFPCR) for amplification of DNA fragments of different lengths. Since thermal isolation of the required temperature zones was essential for optimal biochemical reactions, three finite element models, executed with ANSYS (v. 11.0, Canonsburg, PA), were used to characterize the thermal performance and guide system design: (1) a single device to determine the dimensions of the thermal management structures; (2) a single CFPCR device within an 8 mm × 8 mm area to evaluate the integrity of the thermostatic zones; and (3) a single, straight microchannel representing a single loop of the spiral CFPCR device, accounting for all of the heat transfer modes, to determine whether the PCR cocktail was exposed to the proper temperature cycling. In prior work on larger footprint devices, simple grooves between temperature zones provided sufficient thermal resistance between zones. For the small footprint reactor array, 0.4 mm wide and 1.2 mm high fins were necessary within the groove to cool the PCR cocktail efficiently, with a temperature gradient of 15.8°C/mm, as it flowed from the denaturation zone to the renaturation zone. With temperature tolerance bands of ±2°C defined about the nominal temperatures, more than 72.5% of the microchannel length was located within the desired temperature bands. The residence time of the PCR cocktail in each temperature zone decreased and the transition times between zones increased at higher PCR cocktail flow velocities, leading to less time for the amplification reactions. Experiments demonstrated the performance of the CFPCR devices as a function of flow velocity, fragment length, and copy number. A 99 bp DNA fragment was successfully amplified at flow velocities from 1 mm/s to 3 mm/s, requiring from 8.16 minutes for 20 cycles (24.48 s/cycle) to 2.72 minutes for 20 cycles (8.16 s/cycle), respectively. Yield compared to the same amplification sequence performed using a bench top thermal cycler decreased nonlinearly from 73% (at 1 mm/s) to 13% (at 3 mm/s) with shorter residence time at the optimal temperatures for the reactions due to increased flow rate primarily responsible. Six different DNA fragments with lengths between 99 bp and 997 bp were successfully amplified at 1 mm/s. Repeatable, successful amplification of a 99 bp fragment was achieved with a minimum of 8000 copies of the DNA template. This is the first demonstration and characterization of continuous flow thermal reactors within the 8 mm × 8 mm footprint of a 96-well micro-titer plate and is the smallest continuous flow PCR to date.