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Chapter 18

Integrated Af fi nity and Electrophoresis Systems for Multiplexed Biomarker Analysis

Pamela N. Nge , Jayson V. Pagaduan , Weichun Yang , and Adam T. Woolley

Abstract

The integration of af fi nity columns in micro fl uidic devices generates a micro-total analysis system which has high value in applications such as analyte extraction and preconcentration. In this chapter we describe the preparation of af fi nity columns in situ by photopolymerization of acrylate monomers. The epoxy groups on the columns are further functionalized with antibodies to form af fi nity columns. We describe in detail the use of our af fi nity columns in extracting cancer biomarkers from model mixtures and blood serum. The puri fi ed biomarkers are then eluted from the column, separated by microchip capillary electro-phoresis, and detected by laser-induced fl uorescence. Our procedures allow ef fi cient sample pretreatment and preconcentration, as well as simultaneous and rapid quanti fi cation of multiple biomarkers.

Key words: Capillary electrophoresis , Hot embossing , Microchip , Micro fl uidics , Monolith , Preconcentration , Protein–antibody interaction

Biomarkers can be used in noninvasive early-stage disease detection and in assessing patient response to treatment ( 1 ) . Present methods for biomarker analysis, in prostate cancer, for example, are neither sensitive nor speci fi c enough to enable early-stage detection ( 2 ) , such that improvements are needed. A promising route to probing trace target analytes in complex mixtures entails integrating com-ponents capable of performing sample preparation, separation, and detection into a single device ( 3 ) . Af fi nity columns have strong potential to provide selective analysis of desired components in a complex matrix ( 4, 5 ) . Monolithic supports are an emerging method for chromatographic assays ( 6, 7 ) ; importantly, these

1. Introduction

Terry M. Phillips and Heather Kalish (eds.), Clinical Applications of Capillary Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 919, DOI 10.1007/978-1-62703-029-8_18, © Springer Science+Business Media, LLC 2013

190 P.N. Nge et al.

columns can easily be prepared in micro fl uidic devices by in-situ photopolymerization ( 8 ) . Integration of af fi nity preparation with miniaturized separation offers the advantages of sample extraction and preconcentration coupled with the portability, speed, automa-tion, and reduction of sample volume provided by methods such as microchip electrophoresis ( μ -CE) ( 9, 10 ) . This type of integrated microsystem represents a potential breakthrough in enhancing biomarker determination.

Here we demonstrate two parallel approaches for the prepara-tion of af fi nity columns in poly(methyl methacrylate) (PMMA) micro fl uidic devices. The use of monomers with reactive epoxide groups allows for direct functionalization with antibodies ( 11– 14 ) , which is more straightforward than a multistep process. Application of these columns to biomarker quantitation requires that multiple assay procedures be addressed. First, the biomarkers must be labeled to enable laser-induced fl uorescence detection. Biomarkers also need to be bound to their corresponding antibodies on the column, followed by washing to remove unwanted low-af fi nity components. Moreover, the captured biomarkers must be eluted into a μ -CE system. Finally, the biomarker concentrations are determined either via calibration curve or standard addition methods. These integrated devices offer ef fi cient sample pretreat-ment and preconcentration, as well as simultaneous quanti fi cation of multiple biomarkers in complex mixtures such as blood serum.

Ultrapure water (deionized, 18 M Ω at 25°C) is used to prepare all aqueous solutions.

1. Template: A thermally oxidized silicon á 100 ñ wafer (see Note 1) that has photoresist patterned using photolithography, and which has been etched in 10% buffered HF solution to pat-tern the oxide, followed by wet etching in 40% aqueous KOH solution to yield raised channel features of ~15 μ m height ( 15 ) .

2. PMMA sheets (1.5- and 3-mm thickness) from Cyro Industries, Rockaway, NJ (see Note 2).

3. Glass microscope slides (75 mm × 50 mm × 1 mm). 4. C-clamps. 5. Acetone. 6. Isopropyl alcohol (IPA).

2. Materials

2.1. PMMA Device Fabrication

19118 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

7. Laser cutter 8. Canned compressed gas for dust removal. 9. Copper plates. 10. Precision convection oven.

1. Poly(ethylene glycol) diacrylate (PEGDA, 575 Da average molecular weight).

2. Glycidyl methacrylate (GMA, 97%). 3. Ethylene glycol dimethacrylate (EGDMA, 98%). 4. 2,2-dimethoxy-2-phenylacetophenone (DMPA, 98%). 5. 1-Dodecanol (98%). 6. Cyclohexanol. 7. Tween 20. 8. 0.1 M borate buffer (pH 8.6): Dissolve 3.81 g (10 mmol) of

sodium tetraborate decahydrate in 100 mL water in one fl ask and 0.62 g (10 mmol) of boric acid in 100 mL water in another fl ask. Add the boric acid solution to the tetraborate solution until pH 8.6 is reached.

9. 0.1 M Tris buffer (pH 8.3): Add ~20 mL water to a 100 mL volumetric fl ask. Weigh 0.65 g (5.4 mmol) Tris base and 0.72 g (4.6 mmol) Tris–HCl, and transfer to the fl ask. Mix until the solid dissolves completely. Make up to 100 mL with water (see Note 3). Store at 4°C.

10. 0.1 M carbonate buffer (pH 9.3): Add ~20 mL water to a 100 mL volumetric fl ask. Weigh 0.69 g (8.1 mmol) sodium bicarbonate and 0.20 g (1.9 mmol) anhydrous sodium carbon-ate, and transfer to the fl ask. Mix until the solid dissolves completely. Make up to 100 mL with water (see Note 3). Store at 4°C.

11. 1.5 Dram glass vials. 12. Syringe pump (Harvard Apparatus, Holliston, MA). 13. UV lamps: PRX 1000 (Tamarack Scienti fi c, Corona, CA) and

SunRay (Uvitron International, West Spring fi eld, MA). 14. Black electrical tape. 15. Monoclonal anti-AFP (alpha-fetoprotein) antibody, in phos-

phate buffered saline (PBS) pH 7.2. 16. Monoclonal anti-HSP90 (heat shock protein 90) antibody, in

PBS pH 7.2, containing 50% glycerol and 0.09% sodium azide. 17. Monoclonal anti-CEA (carcinoembryonic antigen) antibody

(produced in mouse), in PBS pH 7.4, containing 0.02% sodium azide.

2.2. Af fi nity Columns

192 P.N. Nge et al.

18. Anti-CytC (cytochrome C) antibody (produced in sheep), in 0.01 M PBS pH 7.4, containing 1% bovine serum albumin (BSA) and 15 mM sodium azide.

1. 0.1 M carbonate buffer (pH 9.3): See Subheading 2.2 , item 10. 2. 0.1 M phosphate buffer (pH 7.0): Add ~20 mL water to a

100 mL volumetric fl ask. Weigh 0.52 g (3.8 mmol) monoso-dium phosphate monohydrate and 0.88 g (6.2 mmol) anhy-drous disodium phosphate, and transfer to the fl ask. Mix until the solid dissolves completely. Make up to 100 mL with water (see Note 3). Store at 4°C. Can be made into PBS by adding 0.88 g (15 mmol) NaCl to make 150 mM once the fl ask is fi lled to the mark.

3. Amicon Ultra-0.5 centrifugal fi lters (30 kDa MWCO). 4. BSA: 10 mg/mL in 20 mM PBS pH 7.0, containing 0.1 mM

EDTA and 5% glycerol. 5. AFP (Lee Biosolutions, St. Louis, MO): 1.75 mg/mL in 0.1 M

PBS pH 7.4, containing 15 mM sodium azide. Exchange this buffer with PBS pH 7.2–7.4 (see Note 4) using an Amicon Ultra-0.5 centrifugal fi lter (see Note 5).

6. HSP90 (Stressgen): 2.1 mg/mL in Dulbecco’s PBS contain-ing 2.7 mM potassium chloride, 1.5 mM potassium phosphate, 137 mM sodium chloride, 8.1 mM sodium phosphate, and 10% glycerol.

7. CEA (Sigma-Aldrich): 1.0 mg/mL in 0.15 M PBS, pH 7.4, containing 0.1% sodium azide (see Note 4).

8. CytC (Sigma-Aldrich): lyophilized powder. 9. Thymidine Kinase 1 (TK1, GenScript, Piscataway, NJ):

0.91 mg/mL in PBS pH 7.4, containing 10% glycerol. 10. Alexa Fluor 488 TFP ester. 11. Fluorescein isothiocyanate. 12. Dimethyl sulfoxide (DMSO). 13. Hydroxypropyl cellulose (HPC, average molecular weight

100 kDa). 14. Serum: Collect blood in a sterile tube without any anticoagu-

lant and let stand for 20–30 min to allow clotting. Centrifuge at 5,000 rpm for 10 min to separate the serum. Remove the serum by carefully pipetting the supernatant off and transfer-ring into a clean vial.

15. High voltage power supplies (Stanford Research Systems, Sunnyvale, CA).

2.3. Biomarker Analysis

19318 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

1. Use the laser cutter to section the 1.5-mm-thick PMMA sheet into 54 mm × 38 mm pieces.

2. For the 3-mm-thick PMMA, use the laser cutter to form 2.5-mm-diameter holes (reservoirs) according to the design in Fig. 1 , and section into 54 mm × 38 mm pieces.

3. Rinse the silicon template with acetone and IPA (see Note 6), and then blow dry with compressed air or nitrogen.

4. Place the silicon template, with the patterned side facing upwards, on a glass microscope slide. Set a 1.5-mm-thick piece of PMMA on the template, with another glass slide atop the PMMA. Sandwich the glass slides with copper plates and hold the assembly together with C-clamps (see Note 7).

3. Methods

3.1. Hot Embossing and Bonding

Fig. 1. Diagram of a micro fl uidic device with integrated af fi nity column. Reservoir labels are A, sample; B, rinse buffer; C, elution solution; D, third standard solution; E, second standard solution; F, fi rst standard solution; G, 5 mM NaOH (to neutralize the acidic elution solution during injection); H, waste; and I–L, electrophoresis buffer. Adapted from ref. ( 11 ) with permission from ACS.

194 P.N. Nge et al.

5. Place the assembly in the convection oven at 140°C for 30 min. The elevated features on the template will be transferred into the PMMA substrate. Remove the assembly from the oven and allow it to cool for a few minutes before removing the clamps and copper plates. Then, place the template against a cooler surface, causing the imprinted PMMA to pull away from the template (see Note 8).

6. Clean both the patterned PMMA and the 3-mm-thick cover plate with canned compressed gas for dust removal and place the cover plate on the imprinted PMMA (see Note 9).

7. Sandwich the two PMMA pieces with glass slides and copper plates and then hold together with C-clamps as in the imprint-ing process (see Note 10).

8. Place the assembly in the convection oven at 110°C for ~25 min to bond the two pieces together (see Note 11).

9. Remove the assembly from the oven and allow it to cool com-pletely before taking off the C-clamps. Then, check to ensure that the device is completely bonded. A schematic of a typical device layout is shown in Fig. 1 .

1. Mix 400 mg 1-dodecanol, 300 mg cyclohexanol, 200 mg GMA, 100 mg EGDMA, and 20 mg DMPA in a glass vial. Vortex the solution brie fl y and sonicate until the DMPA is completely dissolved (see Note 12).

2. Transfer 500 mg of the solution from Subheading 3.2 , step 1 into a clean glass vial, and add 200 mg of Tween 20. Sonicate for 5 min and then purge the solution with N 2 for 5 min (see Note 13).

3. Immediately transfer 5 μ L of the purged solution from Subheading 3.2 , step 2 into reservoir G (Fig. 1 ), and allow capillary fl ow to fi ll the channels in the device (see Note 14); then remove excess solution from the reservoir (see Note 15).

4. Use a photomask, or cover the regions around the af fi nity col-umn with black tape, to make a window for UV exposure. When using black tape, make sure to also cover the backside of the device, except for the window for exposure.

5. Expose the device to UV light for 12–14 min at room tempera-ture using the PRX 1000 lamp (see Note 16).

6. Remove the tape or photomask and immediately apply vacuum to remove unpolymerized solution. Then, fl ow IPA through the channels until the monolithic column becomes white in color (see Note 17).

7. Flush the channels with deionized water until the IPA is com-pletely removed (see Note 18). Then, apply vacuum to remove

3.2. Preparation of Porous Monolithic Columns

19518 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

the water from the device (see Note 19). The monolith can be stored dry at room temperature until it is ready to be function-alized (see Subheading 3.4 ). An electron micrograph of a monolith can be seen in Fig. 2 .

1. Mix 600 mg GMA, 400 mg PEGDA, and 5 mg DMPA (see Note 12). Sonicate the mixture for 1 min and then purge with nitrogen for 3 min to remove dissolved oxygen.

2. Pipette 10 μ L of this mixture into reservoir G (Fig. 1 ) and allow the microchannels to fi ll by capillary action. Then, apply vacuum to reservoir G to suction out most of the monomer solution, leaving a thin coating on the walls of the channel (see Note 20).

3. Cover the microchip with an aluminum photomask having a 4 mm × 4 mm opening to spatially control polymerization, and expose to 200 mW/cm 2 UV light using the SunRay lamp for 5 min (see Note 21). Remove any unpolymerized material by fl ushing IPA through the microchannels using a syringe pump.

1. Dilute the solution of one antibody or a mixture of several antibodies to 0.5 mg/mL in 50 mM borate buffer pH 8.6, pipette into reservoir H (see Fig. 1 ), and let solution fi ll the column (either wall-coated or monolithic) by capillary action (see Note 22).

2. Place borate buffer in all other reservoirs to maintain liquid in the channels during the reaction. Seal the entire chip with clear

3.3. Preparation of Wall-Coated Columns

3.4. Attaching Antibodies to the Columns

Fig. 2. Electron micrograph of a porous monolithic column formed in a micro fl uidic channel. The monolithic structure consists of small globular nodules that offer high surface area, with an average through pore size of ~2 μ m.

196 P.N. Nge et al.

adhesive tape and leave the mixture to react at 37°C for 24 h in the dark.

3. When the reaction is complete, fl ush the device with 100 mM Tris buffer pH 8.3 for 30 min to deactivate any remaining epoxy groups on the column. Then, rinse the entire chip with carbonate buffer pH 9.3 before use.

1. Dilute proteins to 1 mg/mL with carbonate buffer pH 9.3 (see Note 23).

2. For FITC labeling, dissolve 2 mg of FITC in 100 μ L of anhy-drous DMSO. Add 10 μ L of this solution to 250 μ L of protein sample and incubate in the dark for 3 h at room temperature and then overnight at 4°C (see Notes 24 and 25).

3. For labeling with Alexa Fluor 488 TFP ester, dissolve the fl uorescent tag in DMSO to a concentration of 10 mg/mL. Add 5 μ L of this solution to 200 μ L of protein sample and incubate in the dark for 1 h at room temperature.

4. For serum samples, add 2 μ L Alexa Fluor 488 TFP ester in DMSO from Subheading 3.5 , step 3 to 98 μ L of serum sample from Subheading 2.3 , item 14 and label in dark at room temperature for 1 h.

5. Add sodium azide to the labeled proteins to a fi nal concentra-tion of 2 mM. Store the fl uorescently labeled samples in the dark at 4°C until used (see Note 26).

1. Prepare separation buffer by adding HPC to 10 mM carbonate to a fi nal concentration of ~0.5% (see Note 27).

2. Load separation buffer into the channels and all reservoirs except reservoir A (see Fig. 1 ).

3. Place platinum electrodes in reservoirs A, I, J, and L (see Fig. 1 ). Connect the electrodes to the high-voltage power supplies.

4. Perform electrophoresis using “pinched injection” (see Note 28) ( 15, 16 ) .

5. Detect fl uorescence in the separation channel near reservoir L (see Fig. 1 and Note 29).

1. Load separation buffer from Subheading 3.6 , step 1 into the device.

2. Fill reservoirs A–G with the solutions indicated in the Fig. 1 legend and all other reservoirs with separation buffer.

3. Load protein standard on the af fi nity column by applying +600 V for 5 min between reservoir F (or D or E) and reser-voir H (see Fig. 3a ).

3.5. Fluorescence Labeling of Proteins

3.6. Electrophoresis Without Af fi nity Extraction

3.7. Quantitation by Af fi nity Extraction Using a Calibration Curve

19718 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

4. Rinse the af fi nity column with PBS by applying +600 V for 5 min between reservoirs B and H (see Fig. 3b ).

5. Elute the retained analyte and inject by applying +600 V for 1 min to reservoir J while grounding reservoirs C and G (see Fig. 3c and Note 30).

6. Separate the injected standard by applying +2,000 V to reser-voir L, +600 V to reservoirs C and J, and grounding reservoir I (Fig. 3d ).

7. Load sample on the af fi nity column by applying +600 V for 5 min between reservoirs A and H (Fig. 3e ). Then rinse, elute/inject, and separate as for the standards in Subheading 3.7 , steps 4–6.

Fig. 3. Schematic diagram showing quantitation via integrated on-chip af fi nity extraction: ( a ) standard loading, ( b ) rinsing, ( c ) injection, ( d ) separation, and ( e ) sample loading. Adapted from ref. ( 11 ) with permission from ACS.

198 P.N. Nge et al.

8. Plot the peak heights (or areas) from the standard electropherograms against the known protein concentrations. Fit a line to the data via linear regression. Then, calculate the concentrations of the unknown proteins from their corre-sponding peak heights (or areas) in the sample electrophero-gram and their equation of the line.

1. Analyze the sample the same way as for the calibration curve in Subheading 3.7 , step 7.

2. Load sample again on the af fi nity column for 5 min as in Subheading 3.7 , step 7, and then load the fi rst standard on the af fi nity column as in Subheading 3.7 , step 3.

3. Rinse, elute, and inject the material, and then perform micro-chip electrophoresis separation the same as in Subheading 3.7 , steps 4–6.

4. Repeat Subheading 3.8 , steps 2 and 3 for the other two standards.

5. Determine peak heights (or areas) in the electropherogram of the sample and the electropherograms of the sample with dif-ferent amounts of added standard. Plot the peak heights (or areas) vs. the concentration of standard added. Obtain the slope and intercept of the resulting line through least-squares regression. The intercept divided by the slope provides the protein concentration in the sample.

1. Silicon wafers cleave according to their crystalline orientation. To obtain square templates when the wafer is cleaved, silicon á 100 ñ is preferred since it cleaves at 90° angles.

2. PMMA substrates of two thicknesses are used for device fabri-cation. The thinner layer (1.5 mm) facilitates embossing of the pattern from a silicon template. Use of a thicker layer (3.0 mm) for the cover plate enables a larger volume (up to 20 μ L) of solution to fi t into the reservoirs. This ensures that solution does not easily evaporate away during analysis processes.

3. Check the pH and adjust with 1 M HCl or 1 M NaOH if necessary.

4. Buffers containing primary amines such as Tris interfere with the labeling process since they compete for conjugation with the amine-reactive dye. This is also true for buffers containing sodium azide. It is therefore important to exchange such buffers for nonreactive ones like PBS. Other suitable buffers are carbonate and borate.

3.8. Quantitation by Af fi nity Extraction Using the Method of Standard Addition

4. Notes

19918 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

5. This process also concentrates the sample to ~2 mg/mL. Alternatively, dialysis or a desalting column can be used.

6. These solvents remove oil and organic residue from surfaces. Since solvents like acetone can leave a residue on surfaces, a two-solvent method is used.

7. Do not over tighten the clamps as this may break the glass slides or template, causing the hot embossing step to fail.

8. Removing the embossed PMMA when the assembly is totally cooled is not usually a problem. However, detachment after partial cooling prevents melted PMMA from sticking to the sides of the features on the silicon template, which can affect subsequent patterning.

9. This ensures that no particles are trapped in between these plates during bonding, where they could block the channels. Compressed air can also be used, but canned dust removal compressed gas was found to be more effective.

10. The C-clamps should be the same type and size so uniform pressure is applied to the substrates. Uneven pressure can cause one side of the device to be over-bonded with the other side being under-bonded.

11. The time depends on the number of devices being bonded simultaneously. More than two assemblies placed in the oven at the same time may require longer than 25 min.

12. GMA is the monomer; EGDMA (or PEGDA) serves as the crosslinker, and DMPA functions as the photoinitiator.

13. Cap the vial containing the remaining solution and keep it in the dark at room temperature to avoid polymerization. This solution should be used within 10 h. It is necessary to repeat the N 2 purging before fi lling the channel with any stored solution.

14. Allow 2–3 min to ensure that the channels are completely fi lled and that the solution has stopped fl owing. The channels can also be viewed under a microscope for con fi rmation. Polymerization may be incomplete or absent if the solution is still fl owing during UV exposure.

15. This step is important to minimize polymerization in the reser-voir, which will result in a blocked channel.

16. Using a glass mask can reduce spurious polymerization but will also increase the polymerization time. With black tape, 12 min were suf fi cient to polymerize the monolith, but with a photo-mask 14 min were needed. Placing a white object under the device to re fl ect UV light was also helpful in obtaining com-plete polymerization.

17. Removal of unpolymerized solution immediately is essential to prevent further polymerization and potential channel blockage.

200 P.N. Nge et al.

18. Under a microscope, you can observe an IPA–water interface. Flush the channels with deionized water until this interface disappears, indicating that the channels only contain water.

19. The monolith will appear black under optical microscope viewing when the water is removed.

20. This procedure results in a thin coating (~3 μ m) on the chan-nel walls, rather than a porous monolith as in Subheading 3.2 . Though the loading capacity for this column is lower than for a porous monolith, the wall-coated columns do not get clogged during analysis of complex samples such as blood serum.

21. Place the microchip on a copper plate in an icebath during UV exposure. Cooling helps to limit undesired thermal polymer-ization beyond the af fi nity column area.

22. Do not use Tris buffer because it contains amine groups that will react with the epoxy groups on the monolith.

23. All but one of the proteins in Subheading 2.3 label ef fi ciently at pH ~9.3; TK1, with a p I of 8.75, does not. The average number of dye molecules coupled to TK1 was 0.5 at pH 9.3, 1 at pH 9.8, and ~2 at pH 10.6. This indicates that TK1 may not be reactive enough at pH 9.3 for ef fi cient labeling. Higher pHs favor deprotonation of amine groups, resulting in a greater likelihood of amine-reactive dye conjugation to the protein.

24. Discard any unused label, as degradation occurs rapidly once the fl uorophore is dissolved in solvent.

25. Separate unconjugated dye from the protein samples by dia fi ltration, using an Amicon Ultra-0.5 centrifugal fi lter (30 kDa MWCO) and 10 mM PBS pH 7.4. This step is not necessary when af fi nity puri fi cation is done on-chip.

26. For long-term storage, the sample should be divided into small portions before freezing. Then, one aliquot can be removed and used when needed. This avoids repeated freezing and thawing which can break down the sample.

27. HPC is added to suppress electroosmotic fl ow and prevent adsorption of proteins to the channel walls.

28. For pinched injection, reservoirs A, I, and L are grounded while +600 V are applied to reservoir J. For separation, reser-voir I is grounded, +600 V are applied to reservoirs A and J, and +1,600 V are applied to reservoir L.

29. We use an inverted microscope coupled with laser-induced fl uorescence equipment. Brie fl y, a 488 nm laser is focused within the separation channel near reservoir L using a 20× 0.45 NA objective. Fluorescence is collected via the same objective, fi ltered spectrally and spatially, and detected with a photomul-tiplier tube. Detector signal is ampli fi ed, fi ltered, and recorded on a computer. Data points are sampled at 20 Hz.

20118 Integrated Affinity and Electrophoresis Systems for Multiplexed Biomarker Analysis

30. The elution solution in reservoir C is phosphoric acid/dihy-drogen phosphate, pH 2.1. The low pH is needed to disrupt the protein–antibody interaction and elute the protein. Grounding of reservoir G fl ows 5 mM NaOH into the eluting sample and neutralizes the acidic elution solution to ensure optimal separation. Protein samples did not separate well in very acidic buffers.

Acknowledgments

This work was supported by the National Institutes of Health (R01 EB006124).

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