Medical Device Link.

PROCESSING TECHNOLOGIES

Figure 1. (click to enlarge) LabCD cap valve. The two inlet streams entering from the left and right are mixed and then directed out the top channel.

Miniaturization has emerged as a means to address the need for faster analysis of analytes with smaller sample and reagent volumes. Scaling functionality down to the micron and submicron levels was initially developed for microelectronics in the late 1950s. But application to IVD and analytical applications was not realized until the late 1970s, when silicon technology was extended to mechanical microdevices such as pumps, gates, valves, and mixing chambers. Such microdevices became known as microelectromechanical systems, or MEMS.

In the 1980s and 1990s, other components were added to MEMS such as optical and mechanical sensors. New materials, primarily plastics, were developed to decrease production costs and increase ruggedness of MEMS. Such new microdevices depended on microfluidics to move fluids through them in order to perform specific functions.

Microfluidics is a method of moving small volumes (less than 1 ml) of liquid through microstructures such as channels, valves, and gates. Microfluidic devices include fluid control devices, ink-jet printing, gas and fluid measurement devices, and medical devices such as implantable drug pumps. IVD and analytical microfluidic devices include nucleic acid and protein microarrays, and devices designed for specific biochemical and immunoassay applications.

In 2006, the market for all micro­fluidic devices generated approxi­mately $4 billion in revenues. By 2010, this market is predicted to grow to $6.3 billion, with an average annual growth rate of 12%. Sales of microfluidic devices in the medical and life science market were approximately $375 million in 2006. This market segment is projected to grow at an annual rate of 25% to $800 million by 2010. The increased proportion of microfluidic devices in medical applications from 8% in 2006 to 13% in 2010 will be primarily due to the growth of microfluidic diagnostic and analytical applications.

Microfluidics Basics

Microfluidics provides another method for performing molecular diagnostics measurements and assays for clinical diagnosis, drug discovery, screening, environmental analysis, and food testing. The primary distinction is that microfluidic assays require only microliters to nanoliters of sample volume. This distinction can result in the following: conservation of samples and reagents, reduced liquid use, handling, and waste, rapid mixing and reaction (from seconds to minutes), potential for higher assay sensitivity and throughput (e.g., arraying, multiplexing, many samples per assay or many assays per sample), smaller and easier to use systems, and cost savings. However, microfluidics alone cannot increase sensitivity beyond the assay’s inherent sensitivity nor improve the assay of a sample with low analyte concentrations.

Microfluidic devices include the following basic features: channels through which a fluid moves, gates and valves that regulate the direction of flow, and a force or pressure that provides the energy to move the fluid through the channels.

Channel length and width can be varied to act as valves and mixers. For example, if the diameter of a channel gets smaller, more pressure is needed to move the fluid from a larger to a smaller channel.

The two basic types of microfluidic valves are passive and active. Passive microvalves control a liquid’s flow by nonmechanical means, manipulating valve shape and geometry, to slow down or speed up flow.

Figure 2. LabCD serpentine channels for premixing of reagents/ samples prior to reaching a final mixing chamber.

Passive microvalves can also be designed for functions such as reagent mixing. For example, Figure 1 illustrates a cap valve for accepting, mixing, and directing two fluid streams on a LabCD microfluidics disk. Figure 2 illustrates a serpentine channel for passive mixing on the same disc. Serpentine channels can also extend the time it takes for a reagent to reach a reaction chamber.

Active microvalves are pressure-activated mechanical devices that modify the flow of a fluid through the valve. Active microvalves contain components common to all valves: the body, a valve seat to manipulate the fluid (open, closed, or a gradient), and an actuator to control the valve seat’s position. The actuator in an active microvalve is the most critical component that determines the microvalve’s function and reliability. Active microvalves can be digital (only open and closed) or analog (proportional flow control from off to on).

Force is required to move fluid through a microfluidic device. Force can be delivered by using mechanical, magnetic, or electrical pumps.¹ Force can also be delivered by moving fluids through the device using centrifugal force, such as spinning the aparatus. The latter is the basis for LabCD.

Fabricating microfluidic devices utilizes methods such as photolithography, chemical vapor deposition, micromachining, and soft embossing to develop the basic device design. This design can be the basis for developing molds that can be used for production runs applying methods such as injection molding. Injection molding combined with specialized but inexpensive plastics (e.g., polymeth­acrylates, polycarbonate, and poly­sulfone) can produce thousands of devices with high reproducibility at low unit costs in a production run.

Examples of Microfluidic Systems

Table I. (click to enlarge) Examples of microfluidic analysis and diagnostic systems.

Among the first analysis systems using microfluidics were labs-on-a-chip or micro total analysis systems (µTAS). These systems are used for separating and analyzing substances, and can include separation, chemical reaction, detection, and quantification functions (usually external to the microfluidics platform). Specific examples include systems for analyzing amino acids, DNA/RNA extraction and analysis, and protein analysis (see Table I). They can combine methods such as cell extraction, electro­phoresis, chromatography, chemical analysis, enzyme reactions, and end-product detection.

For example, one of the first microfluidic systems for nanoliter DNA sample analysis used an integrated silicone chip.² The device integrates electrophoretic separation, DNA amplification, and fluorescence detection of DNA concentrations as low as 10 ng/ml in 1–2 minutes on a device that is 47 × 5 × 1 mm.

Microfluidic lab-on-a-chip technologies have been applied to various diagnostic and analytical applications. Such applications range from miniaturizing older technologies such as capillary electrophoresis to developing new means for rapid sample analysis in the laboratory (e.g., the GeneXpert system by Cepheid) and point-of-care analysis (e.g., lab cards for cancer and infectious agents by Micronics) (see Table I).

As with any new and emerging technology area, many of the products listed in Table I are still being developed and are not yet available as routine, reproducible assays or methods. However, they represent a new approach to analyzing and diagnosing biological materials which will increase sample throughput in less time and, as they evolve, decrease costs compared with current diagnostic and analytical methods.

Centrifugal Microfluidic Platform

LabCD is a microfluidic reaction platform that uses centrifugal force to move, mix, and react reagents and samples, and to isolate or amplify specific macromolecules such as DNA or proteins. Enzyme reactions and immunoassays produce end products that can be detected and quantified using standard colorimetric or fluorimetric detectors. Isolation or amplification results in end products that can be recovered for further analysis.

In 1996, Gamera Bioscience Corp. (Medford, MA) filed the first patent covering the LabCD centrifugal microfluidic technology, which is protected by more than 60 issued and pending patents.³ The technology was developed to overcome the limitations of microfluidic platforms associated with valving, system integration, performance reproducibility, and high manufacturing costs.

To date, the LabCD platform has been applied to specific molecular analysis and diagnostic systems including the following: nucleic acid amplification (e.g., polymerase chain reaction [PCR]), immunoassays, enzymatic and cell assays, plasmid DNA and protein preparation, diabetes testing, serum protein binding, on-disc hepatocyte drug metabolism assays, and protein crystallization.^4–6 In 2001 and 2003, the first commercialized LabCD products, the LabCD-48 and LabCD-96 ADMET systems for drug discovery, were introduced and provided an automated, rapid platform for assessing the toxicity of drug candidates on cytochrome P450 (CyP) enzymes.⁷

LabCD was the first microfluidic diagnostic platform to use a soft embossing method for initial disc development.⁸ In this method, the original disc design master is tooled out of a polymeric plastic by using a combination of photolithography and computer-controlled machining. A high-durometer silicone rubber is poured into the master to produce a flexible elastomer negative containing all the original microfluidic features. The elastomeric negative produces as many as 50 thermoplastic discs per day using hot embossing.

This approach allows for rapid prototyping and changes in the disc design as the assay on the disc is optimized. Once the design is finalized, metal mold inserts can be assembled to produce hundreds of discs per day using injection molding. For example, the LabCD-96 disc for ADMET assays can be produced by injection molding in daily lots of 500 discs.

A LabCD can be loaded with sample reagents by using either manual multichannel pipettes or automated liquid-handling platforms such as the Tecan Genesis or Gemini Systems by Tecan (Mannedorf, Switzerland). Specialized versions of the latter systems have been modified to allow automated runs of up to 24 discs from initial reagent/sample loading to end product detection and data output.

Figure 3. Tecan Gemini System for LabCD. Discs are loaded by a 8-channel automated pipette and then moved to the incubator (back stacks) or spinner-reader (right) as required for the assay.

Detection of reaction products on LabCD uses a spinner-reader linked to software, which controls various spin rates and times (the spin profile that is developed for each assay). The Tecan reader for LabCD can read either colorimetric or fluorescent assay end products. The spinner-reader can act as a stand-alone instrument or be integrated with the automated liquid-handling workstation. For example, the Genesis LabCD workstation includes an integrated spinner-reader (see Figure 3).

LabCD still needs to be further developed before being widely accepted as a new diagnostic platform technology, in particular the final design and production of discs for generic assays such as immunoassay and nucleic acid isolation and characterization. A simpler, cheaper spinner-reader instrument that is within the cost constraints of clinical and academic labs is also needed.

 

Diagnostic Applications

Table II. (click to enlarge) LabCD assays and methods.

Table II summarizes the assays and methods that have been developed on LabCD. All of them share common operational features including the following:

• On-disc integration of all assay and method steps.
• No moving parts: all valving, mixing, and movement of reagents and samples are driven by programmed steps of increasing centrifugal force.
• Time to reaction product or purified sample of 5–25 minutes for assays, and 10–25 minutes for extraction of DNA or proteins.
• Total reaction volumes of 4–20 µl.
• On-disc quantification of colorimetric or fluorescent reaction products.
• From 12 to 96 simultaneous assays or procedures.
• Inexpensive, plastic, disposable platforms.
• Directly adaptable to automated liquid-handling instruments.
• Common spin-detector unit and software for programming and controlling disc function.

The key to LabCD is eliminating the moving parts. Mechanical gates or valves are unnecessary on LabCD since all movement is controlled through channels and valves of varying diameters. Movement into and through a specific channel or valve is a function of diameter and applied centrifugal force. Accurate volumes (±0.1%) can be delivered to reaction chambers using this principle.

Figure 4. (click to enlarge) LabCD metering valve.

For example, Figure 4 illustrates a LabCD metering valve. At a low, beginning spin speed, the valve fills with reagents. However, at this speed, the liquid cannot move out of the valve through the smaller-diameter channel. Once the excess reagent flushes out of the large feed channel, the spin speed can be increased to force the reagent out of the valve into the reaction chamber.

Figure 5. LabCD-96 disc. While originally developed for ADMET assays, the disc can be used for any assay requiring two reactant solutions.

On the LabCD-96 disc originally developed for the ADMET assays, 192 of the valves enabled two reaction solutions for the 96 assays to be precisely mixed and delivered to the reaction chamber with a 2-minute computer-controlled spin program (see Figure 5). This approach can be applied to any assay in which mixing the components of two solutions (e.g., samples in one and reagents in the other) results in a reaction product such as an antibody-antigen complex, enzymatic degradation product, or receptor-ligand complex.

For example, a LabCD-48 disc that is able to carry out 48 simultaneous, two-chamber assays was used to develop a generic displacement assay system applicable to receptor-ligand and antibody-antigen binding assays.8 The initial target for this assay was the binding of drugs to serum-binding proteins and, specifically, subdomains IIA and IIIA of human serum albumin (HSA) and human alpha-1 acid glycoprotein (ACP). Drug binding to these proteins can alter the pharmacology of the drugs.

It has become a routine diagnostic process in early drug discovery to characterize the binding of new drug entities to these proteins using displacement reactions. The current methods use equilibrium dialysis followed by liquid chromatography/mass spectroscopy, or spectrophotometric or microtiter plate assays.

Figure 6. (click to enlarge) Basis for the LabCD Serum Binding Protein Assay. Displacement of the fluorescent probe by a drug leads to a decrease in fluorescence. The same approach and disc can be used for immunoassay and receptor-ligand assays.

The LabCD method uses dansylated ligands that specifically bind to the subdomains of HSA and ACP, and fluoresce when bound to the protein (see Figure 6). Displacement of the ligand by the drug leads to a loss of fluorescence compared with controls. By reacting a concentration range of drug solutions that are initially loaded into the disc’s sample load chambers with a constant amount of protein-ligand solution, the results are displacement curves and IC50 concentrations at which 50% of the ligand is displaced. The LabCD determined the IC50 values for 21 drugs acting on either HSA or ACP. All of the results agreed with the values found in the microtiter plate assay, with the advantage that the LabCD assays required a reaction volume of less than 20 µl and a total assay time of less than 15 minutes.

Microfluidic Diabetes Panel

Figure 7. (click to enlarge) LabCD Diabetes Panel for simultaneous determination of glucose, total hemoglobin, and glycated hemoglobin in blood.

More-complex LabCDs have been designed for diagnostic assays and genomics. For example, a LabCD diabetes panel was developed for simul­taneously determining glucose, hemoglobin (Hb), and nonglycated hemoglobin (NGHb).⁹ The basic microfluidic disc design for the analysis is shown in Figure 7. All separation and analysis procedures are done on the disc by moving reagents and samples through a series of capillary valves and reservoirs with increasing centrifugal force.

Referring to Figure 7, as the sample enters the disc (a), metered blood samples are taken for Hb (b) and glucose analysis (c, f). Glucose is determined with a standard colorimetric assay such as hexokinase/glucose-6-phosphate dehydrogenase/NADP or glucose oxidase/peroxidase on a nylon matrix pad (g). The result is a colored product proportional to glucose concentration. The reaction product absorbance is read directly from the pad and quantitates glucose present in the fixed volume sampled. The Hb sample is lysed (i–k), and a sample is taken (l–n) for affinity chromatography on a boronate support (o) which retains the glycated Hb. NGHb is washed into a series of cuvettes (r) for colorimetric determination at 430 nm. Total Hb is determined from the lysed sample (m), and glycated Hb can be determined by subtracting the amount of NGHb found from total Hb found.

The entire procedure from sample addition to result takes approximately 15–20 minutes at centrifugal speeds starting at 50 rpm and ending at 3000 rpm. Colorimetric determinations are made with the LabCD spinner-reader. Each disc has four assay arrays enabling four simultaneous blood analyses.

Table III . (click to enlarge) Comparison of the LabCD diabetes panel with commercial diagnostic tests.

The results from the LabCD diabetes panel were compared with commercial IVD tests for glucose and nonglycated hemoglobin (see Table III). For glucose, the results from each method were compared to calculate correlation coefficients. For NGHb, the results from each method were compared with a standard HPLC method for NGHb. The LabCD system showed excellent performance correlations with the commercial systems, in addition to lower assay times and required sample and reagent volumes (i.e., less than 100 µl of blood is added to the disc for analysis).

Focus on Genomics

Figure 8. (click to enlarge) LabCD Plasmid DNA Extraction Disc.

Genomics has been another diagnostic focus in LabCD disc development. Complete on-disc methods have been developed for DNA extraction, PCR amplification, plasmid preparation, and SNP detection (see Table II). For example, Figure 8 illustrates the LabCD disc used for plasmid DNA isolation. A sample of a bacterial culture is moved through a series of channels, metering valves, and filters to lyse and isolate DNA.

Figure 9. (click to enlarge) Gel electrophoresis of plasmid DNA extracted on LabCD compared to a commercial extraction method.

In a series of E. coli experiments, the LabCD disc results were compared with extraction using a Mini plasmid extraction kit by Qiagen (Venlo, The Netherlands). A 5-µl culture sample was applied to the disc, and the various stages of lysis, washing, filtering, and collection of extracted DNA were done with a spin program starting at 300 rpm and ending at 900 rpm over 10 minutes. The resulting DNA was taken from the disc and amplified using PCR with plasmid-specific primers, in parallel with amplification of the Qiagen-extracted DNA. Gel electrophoresis of the amplified DNA showed that the LabCD method matched the Qiagen DNA extraction kit (see Figure 9). The identity of the DNA band on the gels was established by amplifying and running a known (purchased) sample of the plasmid.

Up to four individual plasmid isolation units can be placed on a single LabCD disc. As with the other LabCD assays, the LabCD plasmid disc requires less sample and reagent volumes than those needed for DNA extraction in current methods. This LabCD can also complete the extraction faster, in an automated sample-in, product-out format.

The development of the LabCD plasmid disc and a modified disc for isolation of genomic DNA from blood led to the development of a combined DNA extraction and PCR amplification disc: the LabCD-PCR Plus disc. This LabCD can contain up to 16 DNA extraction and amplification units by using 5–10 µl of sample (e.g., blood, culture, etc.), and can complete the entire process in 20–30 minutes depending on the number of PCR cycles. A number of studies have described the design and function of this disc. ^6,9,10

Samples (5 µl) of a culture or biological fluid are applied to the disc. Using a dedicated spin program, the samples are lysed in 5 µl of 10 mM NaOH at 95°C for 1 minute to release DNA and denature proteins. The DNA solution is neutralized with 5 µl of 16 mM tris-HCl (pH 7.5), and mixed with 8–10 µl of PCR reagents and primers. The DNA is amplified on the disc with thermister heating elements on a spinning platen containing two thermoelectric devices for each assay. A disc can hold up to 16 individual microfluidic structures, allowing for 16 simultaneous amplification reactions.

Figure 10. (click to enlarge) On-disc extraction and amplification of E. coli genomic DNA. The disc-amplified DNA was identical to DNA amplified using a standard thermocycler method.

In one example, the DNA from 2- or 10-µl samples (8 × 103 or 4 × 104 cells) of E. coli were extracted on the disc. PCR amplification was carried out using the lac I primer, which targets a 422-bp sequence encoding the lac I repressor protein, and the EcoCtl primer, which amplifies a randomly selected 300-bp sequence. On-disc amplification used a denaturing step (95°C, 2 minutes) and 35 amplification cycles (anneal at 60°C for 30 seconds, extend at 72°C for 30 seconds, and denature at 95°C for 30 seconds). The amplified DNA was recovered from the disc and analyzed using ethidium bromide–stained agarose gel electrophoresis. The on-disc amplified DNA was comparable to a benchtop amplification using a PTC-100 Thermocycler by MJ Research (Waltham, MA) (see Figure 10).

Conclusion

Richard F. Taylor, PhD, is president of TC Associates Inc. (West Boxford, MA), an independent technical and management consulting firm focusing on diagnostic and pharmaceutical markets. He can be reached at tcadtaylor@cs.com.

Microfluidics offers a new approach for the rapid analysis of clinical and pharmaceutical analytes in a variety of arrayed and multiplexed formats that integrate sample preparation, assay, and end-product detection on the same platform. The low volumes and fast reaction times of microfluidic assays can reduce costs associated with expensive reagents, sample preparation, and assay complexity. The primary challenges for microfluidic assay platforms are manufacturing costs and reproducible performance. The key to overcoming these challenges is developing a design that minimizes inconsistent performance and simplifies mass production.

The LabCD described in this article addresses and meets these challenges by having no moving parts and depending only on centrifugal force to drive the assay reactions. These attributes have also enabled transfer of LabCD designs to mass production by injection molding, producing thousands of reproducible discs in production runs. Linked to an automated liquid-handling and detection system, LabCD has established that microfluidics can be applied to a wide range of IVD and analysis systems.

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