Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling

Abstract

The integration of sample preparation methods into microfluidic devices provides automation necessary for achieving complete micro total analysis systems. We have developed a technique that combines on-chip sample enrichment with fluorescence labeling and purification. Polymer monoliths made from butyl methacrylate were fabricated in cyclic olefin copolymer microdevices and used for solid phase extraction. We studied the retention of fluorophores, amino acids and proteins on these columns. The retained samples were subsequently labeled with both Alexa Fluor 488 and Chromeo P503, and unreacted dye was rinsed off the column before sample elution. Additional purification was obtained from the differential retention of proteins and fluorescent labels. A linear relation between the eluted peak areas and concentrations of on-chip labeled heat shock protein 90 samples demonstrated the utility of this method for on-chip quantitation. Our fast and simple method of simultaneously concentrating and labeling samples on-chip is compatible with miniaturization and desirable for automated analysis.

Figure 1

Schematic of microfluidic devices used for on-chip labeling. a) Simple, two reservoir design used for initial testing. b) Layout used for integrated experiments. The reservoirs are: 1 – sample, 3 – fluorescent dye, 4 – rinse buffer, 5 – eluent, and 2 and 6 – buffer. The lengths from reservoirs 1, 2, 4 and 5 to the injection intersection are all 0.5 cm.

Figure 2

SEM image of a BMA monolith showing detailed morphology.

Figure 3

Normalized retention of a) fluorescent dyes, and b) amino acids and proteins on an on-chip BMA column. Loading was the same for all samples, except the polarity was reversed for Chromeo P503. Values were normalized to 100 nM dye concentration in (a) and 100 ng/mL amino acid or protein concentration in (b).

Figure 4

Elution profiles of fluorescent dyes from an on-chip BMA column. All signals were normalized to 100 nM dye concentration, as in Figure 3. Dyes (from bottom to top) are: Chromeo P503, fluorescein, FITC, and Alexa Fluor 488 TFP ester. Chromatograms are offset vertically for clarity. Positively charged Chromeo P503 dye was eluted with application of a negative voltage, while all others (negatively charged) required a positive voltage. The first 30 s of Chromeo P503 dye elution is expanded in the inset.

Figure 5

Elution profiles of amino acids and two proteins (BSA and HSP90) from an on-chip BMA monolith. a) Chromatograms of amino acids (100 ng/mL) from bottom to top are: aspartic acid, glycine, and phenylalanine. Chromatograms are offset vertically for clarity. b) Elution of 1 μg/mL proteins (BSA – maximum around 55 s, and HSP90 – maximum around 30 s).

Figure 6

Elution profiles of 1 μg/mL BSA labeled a) off-chip and b) on-chip with Chromeo P503 dye.

Figure 7

On-chip labeling and elution of HSP90 using a BMA column. a) Elution traces. Chromatograms are offset vertically for clarity (bottom: 0.1 μg/mL, middle: 0.5 μg/mL and top: 1 μg/mL). b) Plot of peak area as a function of HSP90 concentration. The slope is 7200 ± 140, and the intercept is 830 ± 170.