Journal of Laboratory Automation

Dilution-based volume transfer steps are commonly performed for many types of microtiter plate-based assays. Because the exact concentrations of target samples in solution are typically not directly measured but calculated from the dilution ratio, it is important to accurately measure each dilution step so that the ultimate concentrations of those samples can be known. This paper describes a new approach, which is based on dual-dye, dual-wavelength photometry, for accurately measuring each dilution step in a multistep dilution protocol. The theory behind this method is described, as well as an experimental demonstration of the prescribed approach.

A fully automated electronic microarray control system (Nanochip 400 System) was used to carry out a combinatorial process to determine optimal conditions for fabricating higher order three-dimensional nanoparticle structures. Structures with up to 40 layers of bioderivatized nanoparticles were fabricated on a 400-test site CMOS microarray using the automated Nanochip 400 System. Reconfigurable electric fields produced on the surface of the CMOS microarray device actively transport, concentrate, and promote binding of 40 nm biotin- and streptavidin-derivatized nanoparticles to selected test sites on the microarray surface. The overall fabrication process including nanoparticle reagent delivery to the microarray device, electronic control of the CMOS microarray and the optical/fluorescent detection, and monitoring of nanoparticle layering are entirely controlled by the Nanochip 400 System. The automated nanoparticle layering process takes about 2 minutes per layer, with 10–20 seconds required for the electronic addressing and binding of nanoparticles, and roughly 60 seconds for washing. The addressing and building process is monitored by changes in fluorescence intensity as each nanoparticle layer is deposited. The final multilayered 3D structures are about 2 μm in thickness and 55 μm in diameter. Multilayer nanoparticle structures and control sites on the microarray were verified by SEM analysis.

The use of molecular techniques to inform diagnosis, prognosis, and treatment design will play an important role in the future of medicine. Each new test, however, represents a new cost to the health care system, and significant effort is required to move new techniques to the clinical setting in the most cost-effective and efficient manner. Consequently, there is a compelling need for technological improvements that will facilitate clinical application of novel assays, by reducing cost and complexity in implementation. This is particularly important in cancer pathology. Here, we present a novel applicator technology that enables staining of individual biopsies in a tissue microarray (TMA) to provide low-cost, multiplexed biomarker testing at the level of intact tissue. The applicator is designed to deliver tens of nanoliters of aqueous reagent to arrayed tissue biopsies kept under a layer of oil-based Liquid Coverslip without contacting the biopsies. A pin consisting of concentric stainless steel electrodes separated by a hydrophobic insulator provides a balance between hydrophobicity and hydrophilicity to hold a reagent droplet on the tip of the pin, whereas a small electrical current passed through the droplet spanning the electrodes is used for drop sensing. This design is more amenable to repeatable manufacturing than a previous prototype, which in initial testing demonstrated successful immunohistochemical and in situ hybridization staining of individual biopsies in a TMA, but was difficult to produce. This new design was tested to investigate the factors affecting its operation, in terms of the volume of reagent picked up and its ability to successfully deliver reagent to the biopsies.

Over the past few years, a series of novel microfluidic-based instruments were developed by ThalesNano, Inc. to carry out dangerous and difficult to perform chemical reactions in a safe and fast manner, resulting in superior performance to what commercial batch reactors could provide. Importance of microfluidic devices is continuously raising, as seen there are more and more publications, applications and devices in this field expanding the borders of chemistry. Furthermore, as one of the main advantages for pharmaceutical applications, these new revolutionary reactors allow the fast, on-the-fly mode optimization of different heterogeneous reactions in a high-throughput fashion. The heart of the reactor systems is the actual reactor bed, called the CatCart system. CatCarts allow easy handling of heterogeneous catalyst or immobilized reagents without further purification of products. In addition, the shoe-box size of these reactors makes them available from laboratories to industrial applications.

Drug delivery is still a challenging mission in therapeutic treatment. Research on biomedical micro-electromechanical systems (BioMEMS) has led to a diverse range of microsystems for curative applications. This paper introduces miniaturized controlled valves and drug reservoirs for drug delivery systems. Detailed microfabrication processes, optimized package, and optical/electrochemical detection of the proposed device are described. The release mechanism of the device is controlled by a bilayer actuator valve, which consists of a conductive polymer polypyrrole (PPy) film and a thin metal gold (Au) layer. The PPy layer is electrochemically polymerized on the Au layer. Therefore, further miniaturization of the device is possible through microfabrication of the Au layer. A polydimethylsiloxane (PDMS) package is also introduced to prevent the flap from being blocked by the surrounding tissue of the human body. In addition, a parylene coating is applied to minimize the permeability of PDMS. The release process is then verified by an optical and electrochemical detection system.

In this article, we describe the fabrication and characterization of a reversibly sealed microchip device that is used to couple microdialysis sampling to microchip electrophoresis. The ability to interface microdialysis sampling and microchip electrophoresis in a device that is amenable to reversible sealing is advantageous from a repeated use standpoint. Commercially, available tubing coming from the microdialysis probe is directly inserted into the chip and flow from the probe is interfaced to the electrophoresis portion of the device through integrated pneumatic valves. Fluorescence detection was used to characterize the poly(dimethylsiloxane)-based device in terms of injection reproducibility. It was found that the entire system (microdialysis probe and microchip device) has a concentration response lag time of 170 s. Microdialysis sampling followed by an electrophoretic separation of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide was also demonstrated.

Automated devices and methods for biological sample preparation often use surface functionalized microbeads (superparamagnetic or nonmagnetic) to allow capture, purification, and preconcentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or nonmagnetic functionalized beads that allow samples and reagents to be efficiently perfused over a microcolumn of beads. This approach yields enhanced mass transport and up to fivefold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summary results are given that highlight the analytical performance improvements obtained for automated microbead processing systems using novel microbead trap/flow-cells for various applications including (1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; (2) capture of nucleic acids using oligonucleotide-coupled polystyrene beads with flow cytometry detection; and (3) capture of Escherichia coli 0157:H7 from 50-mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

This review assesses the quality of the data acquired over a 13-week period from a High-Content Analysis screening project that used 297 unique cell lines. This article also evaluates the proficiency of a “tipless” (i.e., does not use disposable tips) full-automation design used for this project that prioritizes intralab system mobility and system configuration mutability. The request to assay a large number of cell lines with poorly characterized growth rates led us to devise an MDS PharmaServices, Inc. proprietary algorithm in an effort to select the proper cell plating density for each cell line. The performance metrics include coefficients of variation (CVs) of Controls for the cell plating data and Data Set Mean CVs for assessing replicate propinquity (i.e., how close the replicates are to each other). The performance of the automation system and our algorithm for this project produced data of superior quality.