Journal of Laboratory Automation

When a gaseous bubble in liquid is excited by acoustic waves, it oscillates (expands and shrinks) at the wave frequencies and generates strong vortical flows around it, the so-called cavitational microstreaming. This article describes the development of a micropumping principle using cavitational microstreaming. The key idea is to place a capillary tube vertically above an oscillating bubble to collect the upward microstreaming flow. When the bubble is excited at its resonance frequency, it oscillates with surface undulations (surface wave mode) and pumps water through the tube. The performance of this pumping mechanism is experimentally studied using millimeter and microscale bubbles. The flow rate and generated pressure are measured in a variety of conditions. The measured results indicate that the present pump falls into the category of moderate-flow-rate and low-pressure type pumps. The present pump operates without physical connections or electrical wiring to the bubbles, implicating potential applications as implantable micropumps in many lab-on-a-chip type systems.

We present a unique immobilization technique via physical adsorption/exchange of biomolecules onto a sensing surface of surface plasmon resonance (SPR). The adsorption/exchange is based on competitive bindings of biomolecules to a surface, which does not require a chemical modification of the biomolecules. SPR sensorgrams show that three human serum proteins, albumin, immunoglobulin G (IgG), and fibrinogen, have different adsorption strengths to the surface and the competitive adsorption of the individuals controls the exchange sequence. A target protein displaces a preadsorbed weak-affinity protein; however, a preadsorbed strong-affinity protein is not displaced by the target protein. In a microfluidic device, we engineer two gold surfaces covered by two known proteins. The sensor allows selective protein detection by being displaced by a target protein on only one of the surfaces. We demonstrate that IgG is selectively detected between albumin- and fibrinogen-adsorbed surfaces. Moreover, the physical adsorption without using an additional surface modification can lead to highly sensitive detection in SPR because of the exponential decay of surface plasmon resonance wave (SPW) from the sensing surface. Based on the competitive adsorption and exchange reaction, we may have a complementary detection system to existing complex and labor-intensive biomolecules detector by bypassing relying on bioreceptors and their attachment to the surface. The physisorbed interface may be useful for automated diagnostic systems.

The development of highly sensitive biosensors for the detection of biomolecules, such as the biomarker interleukin-8 for the early detection of oral cancer, requires optimization of sensor design. To augment the performance of an electrochemical sensor, this study used a microscale, aptamer-based electrochemical sensor for detecting botulinum neurotoxin aptamer hybridization. We first used top—down lithographic processing to define the pattern of the electrodes and then used bottom—up manufacturing to modify the surface molecular properties for reducing nonspecific binding. We systemically examined the effects of the design parameters of an aptamer-based electrochemical sensor. Specifically, five key design parameters were examined: the area of the working electrode (WE), the area of the counter electrode (CE), the separation distance between the WE and CE, the overlap length between the WE and CE, and the aptamer concentration. Through an analysis of the signal and noise generated across variations of the different parameters, the significance of each parameter in sensor performance was determined. In particular, we found that the area of the WE was the only key parameter that influenced the performance of the sensor. The output signal level increased with the area of the WE and the signal-to-noise ratio was about constant in the tested range (i.e., from 0.02 to 4 mm²).

Aptamers are oligonucleotides that can bind to various non-nucleic acid targets, ranging from proteins to small molecules, with a specificity and an affinity comparable with those of antibodies. Most aptamer-based detection strategies require modification on the aptamer, which could lead to a significant loss in its affinity and specificity to the target. Here, we report a generic strategy to design aptamer-based optical probes. An unmodified aptamer specific to the target and a fluorogenic competitor complementary to the aptamer are used for target recognition and signal generation, respectively. The competitor is a hairpin oligonucleotide with a fluorophore attached on one end and a quencher attached on the other. When no target is present, the competitor binds to the aptamer. However, when the target is introduced, the competitor will be displaced from the aptamer by the target, thus resulting in a target-specific decrease in fluorescence signal. Successful application of this strategy to different types of targets (small molecules and proteins) and different types of aptamers (DNA and RNA) has been demonstrated. Furthermore, a thermodynamics-based prediction model was established to further rationalize the optimization process. Because of its rapidness and simplicity, this aptamer-based detection strategy holds great promise in high-throughput applications.

Microfluidic technologies have been applied extensively in rapid sample analysis. Some current challenges for standard microfluidic systems are relatively high detection limits, and reduced resolving power and peak capacity compared with conventional approaches. The integration of multiple functions and components onto a single platform can overcome these separation and detection limitations of microfluidics. Multiplexed systems can greatly increase peak capacity in multidimensional separations and can increase sample throughput by analyzing many samples simultaneously. On-chip sample preparation, including labeling, preconcentration, cleanup, and amplification, can all serve to speed up and automate processes in integrated microfluidic systems. This article summarizes advances in integrated multiprocess microfluidic systems for automated analysis, their benefits, and areas for needed improvement.

An electrowetting-on-dielectric (EWOD) microfluidic system of coplanar electrode type was designed for parallel DNA ligation of an ultra-micro volume, which was applied to droplet manipulation containing DNA, a ligation enzyme and a multi-salt reaction buffer. For environmental reasons, we applied a concept of a water–oil core–shell droplet; such droplets of volume ∼0.3 μL were generated from reservoirs between one plate with coplanar electrodes and another plate with a hydrophobic surface free of electrodes. The experimental results show that the best mixing sequence for the DNA ligation involves mixing first the insert DNA and vector DNA, and then adding the ligase solution. The waiting time was less than 5 min. In one cloning test, the total usage of reagents in an ultramicro-volume EWOD chip was 2.1 μL with no wasted volume, for comparison with 85% waste according to the standard protocol was 15 μL. The results showed also that four parallel DNA ligations were accomplished without damage to a chip and without biomaterial annulment.

A field lab-on-a-chip system was constructed to detect mouse immunoglobulin G (mIgG; model protein target) and Escherichia coli (E. coli; model microorganism target) by using light scattering detection of particle immunoagglutination. The antibodies to these targets were conjugated to the submicron particles by covalent binding, and their long-term stability was evaluated. Antibody-conjugated particles were able to be stored in a 4 °C refrigerator for at least 4 weeks and to be lyophilized as a powder form for the storage in room temperature. The optimum antibody coverage on the particles was 50% for mIgG and 100% for E. coli in terms of assay sensitivity and long-term storage of reagents. Lab-on-a-chip device was fabricated from acrylic plate using an industrial-grade milling machine eliminating the need for photolithography and internal or external pumping. An automatic sampling system was constructed using drip emitters, such that the system can be connected to a pressurized water pipe for detecting E. coli. The automatic sampling system generated the same volume of droplets (70 μL) regardless of pressure. The developed system was successfully tested for E. coli presence in field water samples. The system can potentially be connected to pressurized pipe networks for drinking, processing, irrigation, and wastewater.

A microfluidic system for the enrichment of biological particles, operating on the principle of dielectrophoresis (DEP), is presented. Through the use of a unique manifestation of DEP, contactless DEP (cDEP), this system illustrates the potential to sidestep the common trade-off between sample throughput and selectivity without the need of a complicated fabrication process. The ability to concentrate particles from a sample fluid is validated experimentally through the concentration of 2-μm polystyrene beads and live THP-1 human leukemia cells from a heterogeneous media solution. Finite element analysis of the electric field within the microfluidic channel of the device allows for the determination of effective experimental parameters and accurate predictions of a particle's trajectory through the device. The concentration of particles combined with a fabrication procedure conducive to mass production makes cDEP an attractive alternative to current sample enrichment technologies.

Many components in biological matrices influence the result of an analysis, affecting assay sensitivity and reproducibility. Improved matrix management becomes critical as requirements for higher assay sensitivity and increased process throughput become more demanding. There are several robotic laboratory automation systems that are commercially available, which serve to minimize matrix interference by performing purification and extraction protocols. However, there is an unmet need of inline matrix effect reduction solutions to reduce the processing time and cost for automated sample preparation. In microfluidics, effective matrix management is essential for developing fully integrated systems capable of meeting these requirements. This review surveys current biological matrix management techniques for liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and binding assays with a view toward building automatable processes. For some systems, simple sample-preparation methods, such as dilution and protein precipitation (PPT), are sufficient, whereas other systems require labor-intensive methods, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE). To achieve high throughput, PPT, LLE, and SPE have been adopted to 96-well-plate format. Online SPE has also been coupled with LC-MS/MS to automate sample preparation and analysis of urine, plasma, and serum matrices. However, offline processing of whole blood is still required to obtain plasma and serum. The ultimate goal of implementing sample preparation to reduce matrix effects within untreated sample is to achieve reproducibility and sensitivity required by the application; therefore, inline sample preparation integrated with molecular analysis will be highly significant for laboratory automation. Electrokinetic methods have the potential of handling whole-blood, urine, and saliva samples and can be incorporated into microfluidic systems for full automation. Optimization of analysis conditions and the use of appropriate standards have likewise assisted in reducing or correcting matrix effects and will also be discussed.

With over a decade passed since the first reported use of a Staphylococcal α-hemolysin pore to study single molecules of single-stranded DNA, research in the field of nanopores has advanced rapidly. We discuss the technological progression of nanopore-based devices from the initial use of α-hemolysin pores to the advent of solid-state nanopores to the burgeoning of organic-inorganic hybrid pores driven by the desire to achieve fast and inexpensive DNA sequencing. Additional nanopore-based efforts are also discussed that study other classes of molecules, such as proteins. We discuss the use of nanopores for protein folding and binding analysis. In addition to single-molecule analysis, we report on the introduction of nanopore arrays on thin film membranes for ultrafiltration. Owing to their reduced spatial dimensionality, such membranes offer greater control over how the pores interact with analytes thus leading to very efficient separation. With several technical hindrances yet to be overcome, the devices we report are still works in progress. The realization of these devices will enhance laboratory processes by permitting superior spatial and temporal analytical resolution at the single-molecule level resulting in laboratory capacities of great impact.

Immunoassays have long been widely used in a variety of applications, such as for medical diagnostics, pharmaceutical analysis, environmental, food safety testing, and for basic scientific investigations because of its simplicity, sensitivity, and specificity. Microfluidic systems, also well known as a “lab-on-a-chip” or a “micro-total-analysis-system” have attracted a lot of attention in the past two decades because of advantages associated with miniaturization, integration, and automation. A promising platform for the combination of these two technologies, microfluidic immunoassays, has been extensively explored in recent years. The aim of this article is to review recent advancements in microfluidic immunoassays. A brief introduction to immunoassays and microfluidic devices will include a literature review, followed by an in-depth discussion of essential techniques in designing a microfluidic-based immunoassay from different perspectives, including device substrates, sample/reagent transportation, surface modification, immobilization, and detection schemes. Finally, future perspectives on microfluidic immunoassays will be provided. These developments with microfluidic immunoassays may provide a promising tool for automatic, sensitive, and selective measurements in practical applications.