A butyl methacrylate monolithic column prepared in-situ on a microfluidic chip and its applications.

Abstract

A butyl methacrylate (BMA) monolithic column was polymerized in-situ with UV irradiation in an ultraviolet transparent PDMS micro-channel on a homemade micro-fluidic chip. Under the optimized conditions and using a typical polymerization mixture consisting of 75% porogenic solvents and 25% monomers, the BMA monolithic column was obtained as expected. The BET surface area ratio of the BMA monolithic column was 366 m(2)·g(-1). The corresponding SEM images showed that the monolithic column material polymerized in a glass channel was composed of uniform pores and spherical particles with diameters ranging from 3 to 5 μm. The promethazine-luminal-potassium ferricyanide chemiluminescence system was selected for testing the capability of the column. A flow injection analytical technique-chemiluminescence (FIA-CL) system on the microfluidic chip with a BMA monolithic column pretreatment unit was established to determine promethazine. Trace promethazine was enriched by the BMA monolithic column, with more than a 10-fold average enrichment ratio. The proposed method has a linear response concentration range of 1.0×10(-8) - 1.0×10(-6)g·mL(-1) and the detection limit was 1.6×10(-9)g·mL(-1).

Full text

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Sensors 2009, 9, 3437-3446; doi:10.3390/s90503437

sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
Article
A Butyl Methacrylate Monolithic Column Prepared In-Situ on a
Microfluidic Chip and its Applications

Yi Xu *, Wenpin Zhang, Ping Zeng and Qiang Cao

National Key Disciplines Lab of Novel Micro-Nano Devices and System Technology, and
International R&D Center of Micro-Nano Systems and New Materials Technology, and Microsystem
Research Center, and College of Chemistry and Chemical Engineering, Chongqing University,
Chongqing, 400044, China; E-mails: zhang wenpinchina@yahoo.com.cn (W.-P.Z.);
zp860128@163.com (P. Z.); tianmao6971@163.com (Q. C.)

* Author to whom correspondence should be addressed; E-mail: xuyibbd@sina.com (Y. X.)

Received: 6 February 2009; in revised form: 3 March 2009 / Accepted: 21 April 2009 /
Published: 8 May 2009


Abstract: A butyl methacrylate (BMA) monolithic column was polymerized in-situ with
UV irradiation in an ultraviolet transparent PDMS micro-channel on a homemade micro-
fluidic chip. Under the optimized conditions and using a typical polymerization mixture
consisting of 75% porogenic solvents and 25% monomers, the BMA monolithic column
was obtained as expected. The BET surface area ratio of the BMA monolithic column was
366 m2·g-1. The corresponding SEM images showed that the monolithic column material
polymerized in a glass channel was composed of uniform pores and spherical particles with
diameters ranging from 3 to 5 μm. The promethazine–luminal–potassium ferricyanide
chemiluminescence system was selected for testing the capability of the column. A flow
injection analytical technique–chemiluminescence (FIA–CL) system on the microfluidic
chip with a BMA monolithic column pretreatment unit was established to determine
promethazine. Trace promethazine was enriched by the BMA monolithic column, with
more than a 10-fold average enrichment ratio. The proposed method has a linear
response concentration range of 1.0×10-8 - 1.0×10-6g·mL-1 and the detection limit was
1.6×10-9g·mL-1.

Keywords: BMA monolithic column; separation and preconcentration; microfluidic chip

OPEN ACCESS

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1. Introduction

Sample pretreatment is one of the most important steps in an analytical process. Recently, micro-
fluidic systems have been investigated extensively for biological and chemical analysis because
miniaturization requires smaller samples and offers lower reagent consumption and costs and higher
throughput and performance. However, the capability of microfluidic devices to efficiently handle
complex samples and the integration of sample pretreatment on the same microfluidic chip will be
essential to the successful application of these microfluidic systems. In the Micro Total Analysis
Systems (μ-TAS) field much attention has been paid to sample pretreatment units integrated on
microfluidic chips [1]. To date, solid media have shown special advantages for practical sample
analysis, and some pretreatment methods involving solid media integrated into microfluidic chips have
been investigated recently. Beads are currently used in many analytical systems, although
incorporating them into chips is really difficult [2]. On the other hand, membranes are readily
incorporated into micro-fluidic systems, but have limited applicability due to their small size [3].
Fabricating microstructures on chips is an approach to increasing the surface to volume ratio in a chip
as well as allowing multiple devices to be designed into a chip. This approach has the advantage that
fabrication of multiple supports requires only a little more effort than fabricating a single support.
Finally, monoliths can provide for a solid medium within a channel and can be easily fabricated or
modified to have a wide variety of functionalities [4].
Compared to traditional chromatography columns, the recently developed monolithic columns
prepared in-situ have many advantages such as easy preparation and modification, low operational
pressure, high resolution, large capacity, good permeability, fast mass transfer properties and good
stability [5-7]. These monolithic columns can be used not only as stationary phases for capillary
electro-chromatography and micro-column high performance liquid chromatography (μ-HPLC), but
also as matrices for sample pretreatment and enzyme reactors. Due to their simplicity, speed and
effectiveness, monoliths are especially suited for integration into microfluidic devices, so it is not
surprising that monolithic columns have attracted considerable attention and have been applied widely
in micro-fluidic chip analytical systems in recent years [8]. In this work a butyl methacrylate (BMA)
monolithic column was polymerized in-situ by UV irradiation in a microchannel on a homemade
microfluidic chip for use as a pretreatment device. The fabrication was accomplished successfully and
the resulting device applied to preconcentrate trace promethazine in synthetic plasma samples. It was
thus demonstrated that the butyl methacrylate monolithic column prepared in-situ was highly effective
as a pretreatment unit on a microchip to separate and concentrate some practical samples.

2. Experimental Section

2.1. Chemicals and instruments

A multifunction chemiluminescence analysis system with a PMT detector (MCDR-A, Xi’an Remax
Electronic Co., Ltd.) and syringe pump (Harvard Apparatus, Holliston, MA) was used. Ethylene
dimethacrylate (EDMA) and polydimethylsiloxane (PDMS) were purchased from the Dow Corning
Corporation (Midland, MI, U.S.A). Butyl methacrylate (BMA), 2,2′-azobis-(2-methylpropionitrile)

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(AIBN), potassium ferricyanide, luminol, ammonium acetate, formic acid, acetic acid, ethanol,
methanol, acetone and acetonitrile were acquired from Chongqing Chuandong Chemical Engineering
Reagent Company (Chongqing, P.R. China). γ-MAPS was purchased from the Shanghai Reagent
Company (Shanghai, P.R. China). Promethazine hydrochloride was purchased from the Medicament
Company (Beijing, P.R. China). All aqueous solutions were prepared using double distilled water.
Stock standard solution containing 1×10-3mol·L-l of drug was prepared by dissolving a weighed
amount of promethazine hydrochloride in ammonium acetate (pH=9.3). Standard solutions were
prepared daily by appropriate dilution of the stock solution in ammonium acetate (pH=9.3). The
promethazine stock solution was diluted in a freshly human serum sample to a concentration of
2.0×10-9 g·mL-l. A 1×10-2 mol·L-l stock solution of potassium ferricyanide was prepared by dissolving
the required amount of compound in distilled water. Diluted solutions of potassium ferricyanide were
prepared by mixing portions of the stock solution with the required amounts of NaOH/NaHCO3
solution.

2.2. Microchip Fabrication

PDMS is useful for microfluidic fabrication and application because of several important properties,
including its mechanical flexibility, gas permeability and optical transparency. Moreover, its elasticity
and hermetic self-sealing properties make multilayer construction of PDMS-based devices relatively
straightforward. PDMS molding is used almost exclusively for rapid prototyping in corporate
environments because of its simplicity and fast turnaround time. In this paper, the microchips
employed in the experiments were fabricated with PDMS and glass using standard techniques. The
PDMS chip is cured, removed, and then pressed or bonded onto a glass substrate to create a complex
microchannel by the monolithic pouring method. The bottom chip was fabricated in glass using
conventional wet chemical etching. In a final step, a glass chip was bonded to the PDMS chip to yield
a network of closed channels. The upper layer and the bottom layer having different channels. The
upper layer had the preconcentration microchannels (AB, 5 mm length and 150 μm diameter) and the
lower PDMS layer had “Y” shape reaction microchannels (150 μm wide and 150 μm deep). The
reservoirs (R1, R2 R3 and R4) were punched on the upper PDMS layer using a round hollow punch,

2.3. Monolith column polymerization

For the polymerization of the monolith column butyl methacrylate (BMA), ethylene dimethacrylate
(EDMA), 2,2′-azobis-(2-methylpropionitrile) and methanol/ethanol were used as the monomer, cross-
linking agent, initiator and porogenic solvents, respectively. The detailed monolith column
polymerization processes was carried out as follows: first, the microchannels flushed by sodium
hydroxide solution and double distilled water were washed with silanization solution containing 30%
(V/V) γ-MAPS in acetone. Prior to mixing with the porogenic solvents, EDMA and BMA were mixed
with fresh basic alumina powder to remove the added inhibitor. After purging with nitrogen for 3 min,
the mixture consisting of 75% porogenic solvents and 25% monomers were pumped into microchannel
for the subsequent polymerization. The porogenic solvent used was a mixture of methanol and ethanol
with a ratio of 5:3. A 1:1 mixture of BMA and EDMA was used as monomer. Then, 365 nm UV light

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was used to irradiate the mixture from the top from a 5 cm distance through a mask to control the
location of the BMA monolithic column on the microchip. The BMA monolithic column was
polymerized in-situ in an ultraviolet transparent PDMS microchannel on a homemade microfluidic
chip; it was finally washed with methanol at 2.0 μL·min-1 for 30 min. The monolithic column was then
attached to the FIA-CL micro-syringe pump to separate and preconcentrate trace promethazine in the
synthetic plasma samples.

2.4. Sample analysis process by microfluidic chip

2.4.1. Sample analysis system

The schematic diagram of the microfluidic chip analytical system is shown in Figure 1. The whole
analysis system consist of microfluidic chip, monolithic column, photomultiplier, computer and
syringe pump. In this system, the reservoirs R1, R2 and R3 were connected to microsyringe pumps, R1
for sample solution while R2 and R3 for chemiluminescence reagent and R4 for waste water. In such
configuration, a sample was enriched by the pretreatment monolithic column when the sample was
loaded to the microchannels in the microchip with a microsyringe pump. The enriched sample was
washed with a suitable solution, and then the eluted solutions were injected into the luminol mixture
solution. All the solutions were mixed in a microchannel just before the detection zone in the
homemade microfluidic chip. The intensity of the emission response was obtained in this way by
photomultiplier tube (PMT) detector in a dark housing.

Figure 1. Schematic diagram of the FIA-CL system based on the homemade microfluidic
chip with monolithic column

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2.4.2. Sample loading and elution

The BMA monolithic column on the microchip was washed and preconditioned as follows: after
washing with acetonitrile for 10 min at 3.0 μL·min-1, the monolithic column was preconditioned with
10 mM ammonium acetate buffer (pH 9.3) for 10 min at the same flow rate. The promethazine stock
solution was diluted in ammonium acetate (pH = 9.3) to a final concentration of 2x10-9 g·mL-1, and a
volume of 30 μL of the sample was loaded to the BMA monolithic column at a flow rate of 0.2
μL·min-1. After washing the column with ammonium acetate (pH = 9.3) at a flow rate of 2.0 μL·min-1
for 10 min, the retained promethazine was eluted with acetonitrile containing 0.1% formic acid, which
was pumped into the reaction channel in the glass layer at a flow rate of 0.5 mL·min-1.The
promethazine eluted solutions were injected into the mixture of luminol solution (1.0×10-4 mol·L-1)
and potassium ferricyanide (2.5×10-4 mol·L-1) under basic conditions of NaOH/NaHCO3
(CNaOH:CNaHCO3 = 7:5). The CL intensity was measured by photomultiplier tube. The methodology of
promethazine detection was developed by the chemiluminescence reaction of promethazine with
luminol and ferricyanide in basic medium.

3. Results and Discussion

3.1. Microchip Fabrication technology

In PDMS fabrication technology, the process of creating microfluidic channels in PDMS using SU-
8 as a mold is complicated and very difficult to carry out in an ordinary laboratory. On the other hand,
the monolithic PDMS pouring method offers simpler fabrication procedures, lower costs, faster
turnaround time and higher integration and mechanical performance etc; in addition, the integrated
microchannel can be used repeatedly and it avoids the problem of leakage, which make it useful for
biological and chemical analysis in a microfluidic chip analysis system. In this work, highly
hydrophobic and surface polished copper/platinum wire was used as the channel moulding material in
order to minimize the channel distortion and surface roughness. The size of microfluidic channels
could be controlled by simply changing the size of the copper/platinum wire used. With all this in
mind, the PDMS microchips used in the experiment were fabricated by the monolithic pouring method
as follows: the PDMS oligomer and crosslinking prepolymer of PDMS agent is mixed in a mass ratio
of 10:1. A convenient way of mixing these agents is to place both in a disposable plastic bag, heat seal,
and mix manually. The mixture can be placed under vacuum for degassing. The PDMS mixture is
poured onto the homemade wire shaped mold and cured for 4 h at 60°C (see Figure 2.3). After cooling,
the PDMS can be carefully peeled off the mold (see Figure 2.4). Debris on the PDMS surface must be
removed after punching the holes because the presence of small particles can be detrimental to the
bonding of PDMS to glass substrate. This process yields a PDMS polymer sheet with whole
microchannels. A schematic of the microchip described in this paper is shown in Figure 2. The length
of channels is 20 mm, and the diameter of the channel was 100 μm. The bottom chip was fabricated in
glass using conventional wet chemical etching as described in Figure 2. The detailed microfluidic
channel fabrication process was as follows: firstly, a photomask is required to prepare the
microchannel by computer aided design (CAD) program. A glass substrate with a chrome-gold layer