A Microfluidics-Based Droplet Plate Reader for Nucleic Acid and Protein Quantification

Life science laboratories are gradually moving toward automated liquid handling to achieve cost reduction, productivity increases, and improved data consistency and quality. As a result, scientists can accomplish their research without performing tedious repetitive tasks and at a fraction of the time previously required. A similar trend, focusing on cost reduction and increased assay speed, is the downscaling of sample volumes to microliter levels typically stored in a 96- or 384-multiple-well plate format. A good example can be found in molecular diagnostics. As the demand for molecular tests increases, automated methods for biomolecule isolation are being developed that use a 12- to 96-well format. This approach facilitates integration of the complete molecular test in an automated setup, which includes robotic liquid handlers and bar-coded sample tracking, resulting in significantly higher sample throughput. The method is also less labor intensive and eliminates human error while generating small DNA/RNA or protein samples.

Although biomolecule isolation procedures can now be accelerated and automated, a downstream bottleneck occurs in the quality and yield determination of the extracted samples. For biomolecule quantification, droplet spectrophotometers are widely used due to their simplicity, low-volume sample consumption, and wide linear detection range. However, these instruments are labor intensive and cannot be integrated into liquid handling devices for the automated processing of large numbers of samples. In contrast, UVVIS absorbance plate readers are suitable for high-throughput biomolecule quantification, but require larger sample volumes for accurate measurements and often additional dilution steps to ensure operation within the linear range. Quantification using intercalating fluorochromes offers minimal sample consumption and automation by using fluorometric plate readers. Although the method is well-accepted in high-throughput molecular laboratories, it is costly and requires additional handling steps and reference samples (standard curve).

A new generation of optical instruments and Society for Biomolecular Sciences (SBS)-standard compatible microfluidic consumables combine the advantages of droplet spectrophotometry with the throughput of plate readers. This solution enables direct quantification of small samples in a high-throughput, automated work flow.

Technology

Trinean technology (Trinean, Gentbrugge, Belgium) is based on the DropSense96 multichannel spectrophotometer combined with the microfluidic DropPlate16/96 consumable for storage and UV-VIS spectral analysis of 1–3 μL samples. PC-based DropQuant software allows for simple experimental setup and export of measurement results.

Figure 1 - Trinean technology: a) DropSense96 spectrophotometer and microfluidic DropPlate. b) Description of the filling steps of the microfluidic structures in the DropPlates.

The following microfluidic DropPlate consumables can be read on the DropSense 96 spectrophotometer: the small DropPlate16 has 16 input wells for a low- to medium-throughput mode, and the 96-well microtiter-plate-sized DropPlate96 is for high-throughput use. Both are compatible with liquid handling robots (SBS standards) and permit easy loading and preservation of samples and measurement of the optical absorption of the droplet with variable pathlength.

A typical DropPlate microfluidic structure consists of an input well connected to a capillary storage channel and one or two microcuvettes, as shown in Figure 1. When dispensing a sample droplet into the input well, it is instantly drawn into the storage channel through capillary forces in order to strongly suppress sample evaporation. This allows the user to perform the measurement within a time span of 2 hr after dispensing. After inserting the DropPlate into the DropSense96, pressure-driven transport of the samples to the microcuvettes occurs while simultaneous absorbance measurements are performed to monitor the filling behavior of the microcuvettes and analyze the spectral absorbance of the sample. With the DropPlate16/96D, which has two superposed microcuvettes, a dual-pathlength measurement (0.2 and 1.0 mm) is done, which generates a large OD measurement range (0.1–70 OD, 10 mm equivalent absorbance), thereby eliminating the need for sample dilution. The DropPlate16/96S contains a single microcuvette with an intermediate pathlength of 0.5 mm, which is suitable for the high-speed analysis of samples with a more limited concentration range (0.2–24 OD, 10 mm equivalent absorbance).

The DropSense96 contains four polychromatic spectrometers to capture the full UV-VIS spectrum of four samples simultaneously (no wavelength scanning needed). The spectral analysis of an entire DropPlate96 is performed in about 5 min. After the measurements, the DropQuant software displays the complete UV-VIS absorption spectrum of the samples and stores the data on the connected PC. An additional software library is available for full integration of the DropSense96 system in automated laboratory setups, and the user software is compatible with the data formats most frequently used in databases or LIMS (i.e., xls, csv, txt, and pdf).

Overall, the Trinean technology provides fast and steady measuring conditions as a result of the full UV-VIS range analysis; the fixed pathlengths and flat optical surfaces within the DropPlate; and elimination of solvent evaporation, leading to enhanced reproducibility.

Applications

Based on the flexibility of the consumable selected (DropPlate or standard 96-well plate), complete UV-VIS spectral analysis, standard curve, and kinetic measurements, Trinean technology can be used in a wide range of applications. Predefined functions are available for DNA/RNA (A260) quantification, protein (A280) concentration analysis, determination of labeling efficiency, and colorimetric assay readout. The following describes the performance of the DropSense96 in terms of linearity and reproducibility for dsDNA (A260), protein samples (A280), and colorimetric protein assay readout.

Figure 2 - a) Dilution series of calf thymus dsDNA measured with the DropSense96 and a reference spectrophotometer. The average and variation of five replicates of each dilution are shown. b) Set of calf thymus dsDNA dilutions measured 10 consecutive times; reproducibility was assessed by calculating the coefficient of variation. c) Linear plot of the DropSense96 relative to measurements made with the reference spectrophotometer. d) Typical spectral view of DNA samples with the DropSense96; one replicate of 15, 60, 121, 500, and 2382 ng/μL calf thymus dsDNA is shown.

Nucleic acid samples can be quantified directly using the DropSense96 spectrophotometer by measuring their optical absorption at 260 nm. Furthermore, the full absorbance spectral analysis helps to identify impurities since the 260/230 and 260/280 ratios can be deduced and background levels can be detected in the 320–750 nm region. Measurement accuracy across the dynami c range of the DropSense96 was evaluated by measuring a dilution series of calf thymus dsDNA (Invitrogen, Carlsbad, CA) between 3 and 3500 ng/μL with the DropSense96 using 3-μL samples in DropPlates16D and with a reference droplet spectrophotometer. Each dilution was measured five times, and a comparison of the averages showed very good linear correlation between both instruments since the calculated coefficient of determination (R2) is approximately 1 (Figure 2). The reproducibility of the DropSense96 was assessed by measuring a set of calf thymus dsDNA dilutions in 10 consecutive measurements. All measurements were performed on the same DropSense96 using a random set of DropPlates16D filled with 3-μL samples. As shown in Figure 2, the calculated coefficients of variation (CV) were 2.9, 0.6, and 2.2 at a low concentration of 15.5 up to 121.2 and 1039.7 ng/μL dsDNA.

Figure 3 - a) Dilution series of bovine serum IgG protein measured with the DropSense96 and a reference spectrophotometer. The average and variation of five replicates of each dilution are shown. b) Linear plot of the DropSense96 relative to measurements made with the reference spectrophotometer. c) Typical spectral view of protein samples with the DropSense96; one replicate of 3.0, 6.9, 14.8, 33.1, 44.3, and 62.5 bovine serum IgG is shown.

The concentration of purified proteins, analogous to nucleic acids, can be determined with the DropSense96 by measuring their UV absorbance at 280 nm and calculating the concentration using their extinction coefficient in the Beer-Lambert equation. Because the 280-nm absorption of proteins depends on the presence of aromatic amino acids, the absorption strength of the proteins may vary depending on the particular amino acid content of a protein. To demonstrate the linear range of the DropSense96 for protein quantification, a broad IgG protein (from bovine serum, Sigma-Aldrich, St. Louis, MO) dilution series from 0.2 to 60 mg/mL was prepared and measured with the DropSense96 using 3-μL samples in DropPlates16D and a reference droplet spectrophotometer. Each dilution was measured five times and the calculated averages were determined. Data comparison between both instruments shows good linear correlation with an R2 of 0.9986 (Figure 3).

Figure 4 - a) Dilution series of BSA was used in a Pierce 660-nm colorimetric protein microplate assay and absorption at 660 nm was measured with the DropSense96. b) Linear plot of the DropSense96 relative to measurements made with the reference spectrophotometer. c) Typical spectral view of protein assay standard curve measurements with the DropSense96; one replicate of the Pierce 660-nm assay with 0, 0.5, 1.5, and 4 mg/mL BSA is shown.

Different colorimetric protein assays have been developed as an alternative to the quantification of proteins. The most commonly used methods are the Bradford assay, the Lowry assay, the bicinchoninic acid (BCA) assay, and the Pierce 660-nm assay (Thermo Fisher Scientific, Rockford, IL). The latter is based on the binding of a proprietary dye–metal complex to protein under acidic conditions, which causes a shift in the dye’s absorption maximum, measured at 660 nm. After creating a standard curve of protein solutions with known concentrations, the protein concentration of unknown samples can be calculated (Figure 4). The dynamic range of the 660-nm assay with the DropSense96 was determined using DropPlates16D. The dilution series of bovine serum albumin (BSA) protein (Invitrogen) of 0.05–4 mg/mL was made, and 10 μL of each dilution was mixed with 150 μL reagent in a multiple-well plate. After incubating for 5 min at room temperature, the Drop-Plates16D were loaded with 3 μL sample and measured on the DropSense96. As shown in Figure 4, a BSA concentration range from 0.05 to 2 mg/ mL can be measured with the 660-nm assay using a 1:5 protein:reagent ratio.

Conclusion

Trinean technology, combining a high-throughput spectrophotometer and microfluidic DropPlate consumables, provides highly accurate and reliable absorption data over a wide measurement range. The system offers fast and steady measurement conditions due to the full UV-VIS range analysis; fixed pathlengths; and flat optical surfaces within the DropPlate, while eliminating excessive evaporation for enhanced reproducibility. The system provides a combination of microliter sample readout and high throughput capabilities, which not only minimize sample consumption but reduce sample quantification and quality analysis time by almost 50%. The DropSense96 can serve as a powerful tool in high-throughput laboratories engaged in nucleic acid and protein-based research. The technology places high-speed measurements using a minimal amount of sample within the reach of every laboratory.

Dr. Montoye is Application Developer, Trinean, Dulle Grietlaan 17/3 9050 Gentbrugge, Belgium; tel.: +32 92727535; fax: +32 92727539; e-mail: info@trinean.com.

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