Straightforward and Ultrastable Surface Modification of Microfluidic

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Straightforward and ultrastable surface modification of microfluidic chips with norepinephrine bitartrate improves performance in immunoassays Haiying Shen, Feng Qu, Yong Xia, and Xingyu Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05186 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Analytical Chemistry

Straightforward and ultrastable surface modification of microfluidic chips with norepinephrine bitartrate improves performance in immunoassays Haiying Shen,†,‡ Feng Qu,*,† Yong Xia,*,§ Xingyu Jiang*,‡,§,‖ † School of Life Science, Beijing Institute of Technology, Beijing 100081, People's Republic of China ‡ Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China § Department of Clinical Laboratory, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, People's Republic of China ‖ University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China ABSTRACT: Polymers are commonly used materials for microfluidic chip fabrication because they are standardized in fabrication and low in cost. However, most of polymeric materials that are readily fabricated in industrial scale are hydrophobic, which is inconvenient for the injection and flow of the aqueous solution to result in poor analytical performance for biochemical assays. In this work, we present a straightforward and ultrastable surface modification process for polymeric chips. A one-step modification by using norepinephrine bitartrate monohydrate as modification reagent is completed at room temperature. The hydrophilicity of the polymeric surfaces increases dramatically. Surface modification is stable for at least 2.5 years to allow auto-injection of aqueous solution into the channels. The chips are applied in the immunoassay of alpha-fetoprotein (AFP). The low non-specific adsorption after modification results in significantly decreased background noise, optimized signal-to-noise ratios (SNR) and dramatically enhanced reproducibility of the immunoassay. Thirty clinical human serum samples are analyzed; these results strongly correlated with the values obtained using commercial test kits. We anticipate that this surface modification method can be used for immunoassay devices in analytical and biosensing technology.

INTRODUCTION Over the past three decades, extensive research in microfluidic system has led to significant progress in the development of chemistry, biology and medicine.1-3 Microfluidic technologies can accurately handle fluids in a very small space and make the chemical or biological reaction more rapid and efficient.4-11 For single-use disposable devices, polymeric microfluidic chip is more suitable than those fabricated and chemical etched from glass and silicon,12-15 since it has broader choices in standardized and low cost fabrication methods, such as laser ablation, injection molding, and hot embossing.16-18 Researchers have already used many kinds of polymeric materials such as cycloolefin copolymeric (COC), polycarbonate (PC), polymethylmethacrylate (PMMA), and polystyrene (PS) in the fabrication of microfluidic chips.19-22 Their hydrophobic nature, however, not only prevents aqueous solutions from filling channels, but also causes chips to foul through nonspecific adsorption of biomolecules which results in poor analytical performance. Thus, researchers usually have to use pump system to help injecting and flowing, which makes the whole device complex and inconvenient in point-of-care test (POCT) application. So to modify channels with highly hydrophilic surface will benefit auto-injection and easy cleaning as well as the whole simpler device. Scientists have reported some physical or chemical methods to improve the hydrophilicity of polymeric surface,23-27 but most of their methods

use harsh conditions or complicated processes, also the surfaces are easy to lose their effectiveness over time.28-30 In this work, we present a straightforward and ultrastable surface modification process of polymeric microfluidic chips, which need only one-step modification by using norepinephrine bitartrate monohydrate as surface modification reagent. The modified channels allow auto-injection of aqueous solutions and show good performance in liquid flowing. Moreover, the flow velocity of aqueous solution in the channels modified 2.5 years ago is similar to the newly modified, which indicates that the surface modified by this method can keep hydrophilic ultra-long-term more than twice as the method reported before.23 Furthermore, the AFP detection results of clinical serum samples show that the reproducibility are dramatically improved. EXPERIMENTAL Materials. COC, PC, PMMA, and PS are from Yongqing plastics plant (Hebei, China). Norepinephrine bitartrate monohydrate, tris(hydroxymethyl)aminomethane, alcohol, and hydrochloric acid are from Aladdin (USA). Phosphate buffer saline (PBS) and bovine serum albumin (BSA) are from Merck (Darmstadt, Germany). AFP standard substances, mouse monoclonal AFP antibody, and horseradish peroxidase (HRP)-labeled mouse monoclonal AFP antibody are from KeYueZhongKai Biotechnology Co., Ltd. (Beijing, China). The PDMS base and curing agent (Sylgard 184) are from Dow

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Corning Inc. (USA). The water used in the experiments is purified by using a water purification system (Millipore, USA). Clinical human serum samples are from the 306th Hospital of PLA (Beijing, China). Design and Fabrication of Microfluidic Chips. The microfluidic chips are designed by Unigraphics NX 3D computer aided design software (Munich, Germany). There are two types of the microfluidic chips used in the assays. One is for observing the flow characteristics of the single channel, (Figure S1A) and the other is for array immunoassay (Figure S1B). The single channel microfluidic chip is composed of two layers: the top PMMA fluidic layer and the bottom PDMS substrate layer. The top PMMA fluidic layer contains the channel with the inlet (0.5 mm in diameter) and the outlet (0.5 mm in diameter). The top PMMA fluidic layers are further divided into two types according to different experiments. To observe the flow characteristics in the channels with different widths, the channels are in same height (300 µm), same length (100 mm) and different widths (300, 400, 500, 600, and 700 µm), and the top PMMA fluidic layer is 18 mm in thickness,118 mm in length, and 2 mm in height. To observe the flow characteristics in the chips with different thicknesses, the channels are 300 µm in height, 100 mm in length, 600 µm in width, and the top PMMA fluidic layer is 18 mm in width, 118 mm in length, and different thicknesses (2, 3, 4, 5, and 6 mm). The bottom PDMS substrate layer is a flat sheet which is 18 mm in width, 0.5 mm in thicknesses and 118 mm in length. To form a chip with an enclosed channel, the bottom PDMS sheet is fitted tightly to the top PMMA fluidic layer. The surface of the PMMA layer is smooth enough to combine tightly with the PDMS layer if we make sure that both surfaces are very clean. If there is no conformal contact between these two layers, when we introduce liquids into the microfluidic channels the two layers will separate. The microfluidic chip for array immunoassay is 20 mm in width, 2.85 mm in thicknesses and 55 mm in length. It is also composed of two layers: the top PMMA fluidic layer and the bottom PDMS substrate layer. The top PMMA fluidic layer (20 mm in width, 2.35 mm in thicknesses and 50 mm in length) contains seven parallel channels (500 µm in width, 500 µm in height and 40 mm in length) with the inlet (0.5 mm in diameter), and the outlet (0.5 mm in diameter). The distance between two adjacent channels is 2 mm. The bottom PDMS substrate layer is a flat sheet which is 20 mm in width, 0.5 mm in thicknesses and 55 mm in length. The bottom PDMS sheet is fitted tightly to the top PMMA fluidic layer to form a chip with seven parallel channels. In this study, all the PMMA fluidic layers are manufactured by a computer numerical control (CNC) engraving and milling machine (The Technical Institute of Physics and Chemistry of the Chinese Academy of Sciences, China) and cleaned with alcohol. To make the PDMS substrate layer, we mix PDMS base and curing agent thoroughly with a mass ratio of 10:1. The mixture is degassed under vacuum, poured onto a plate, and cured in a vacuum oven at 70 oC for 60 min. Then we peel the cured PDMS substrate layer from the plate and tailor them to certain size for use. One Step of Modification of Poly-norepinephrine Bitartrate on Polymeric Surfaces. We dissolve norepinephrine bitartrate in tris-HCl buffer (0.05 mol L-1, pH 8.5). We introduce the solution (20 mg mL-1) on the slabs or into the channels immediately and keep it for 2 hours at room temperature.

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We clean the slabs and channels with water. We put them into zip lock bags and store them in 4 oC in the refrigerator. Surface Characterization by Contact Angle Measurements. The hydrophilicity of the polymer surfaces is evaluated based on static contact angle measurement of an aqueous liquid drop on the solid surface. Its hydrophilic property is quantified via the Young equation. The contact angle is measured using the drop shape analyzer (DSA100, Krüss, Germany). In the assay, we introduce 3 µL of 18.2 MΩ cm-1 water onto the COC, PC, PMMA, and PS slabs (manufactured and polished by the Technical Institute of Physics and Chemistry of the Chinese Academy of Sciences) respectively and record the contact angles. The average of measurements taken from 10 replicate tests is reported as the mean contact angle for the modified polymeric slabs. In order to evaluate the stability of the modification, we monitor the contact angles of the modified COC, PC, PMMA, and PS slabs during ten consecutive weeks with the same method above. Also, we use a micropipette to introduce a droplet with red-colored food dye onto the inlet of the microfluidic chip to observe its flow in the modified channel, and record the auto-injection process with the camera (Nikon D90, Japan). AFP Detection Using the Modified Microfluidic Chips. We carry out the AFP detection on the microfluidic chips in the double antibody sandwich format. To immobilize mouse monoclonal AFP antibody stripes onto the bottom PDMS substrate layer of the microfluidic chip, we introduce AFP capture antibody solution (5.0 µg mL−1) into the parallel channels of the top PMMA layer and incubate for 30 min, then suck out the solution and peel off the top PMMA layer. We place the top PMMA fluidic layer which the channels are modified with norepinephrine bitartrate on the bottom PDMS substrate layer. The channels are perpendicular to the mouse monoclonal AFP antibody stripes. For blocking, we introduce 5 % (m/V) BSA solution onto the inlet of the channel, and the droplet flows into the channel automatically. After blocking for 30 min, we suck out the BSA solution and introduce AFP standard solution (1 ng mL-1, 3 ng mL-1, 5 ng mL-1) into different channels and keep 30 min for incubation. The antigen–antibody complexes are formed at the intersection of the channels and the AFP capture antibody stripes. We introduce PBS into channels for cleaning. We introduce HRP-labeled mouse monoclonal AFP antibody solution (5.0 µg mL−1) into the channels and the antibody-antigen-antibody sandwich complex is formed. After incubation for 30 min, we introduce PBS to clean the channels, and peel off the top fluidic layer. We introduce the chemiluminescent substrate onto the bottom PDMS substrate layer and capture the chemiluminescence images using a portable chemiluminiscence imager we designed.5 We use ImageJ software (National Institutes of Health, USA) to analyze the images. In the assay, we used the unmodified channel for control. Detection of Clinical Human Serum Samples and Comparisons with Commercial Products. The serum samples collected from the 306th Hospital of PLA (Beijing, China), are prepared by centrifugation (6000 r min-1, 30min) at room temperature for 10 min after resting the whole blood. We evaluate the intra-assay coefficient of variation (CV) and inter-assay CV of immunoassays using clinical human serum samples by the modified chips. In the intra-assay, we analyzed the same serum sample simultaneously through 49 parallel measure-


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Analytical Chemistry ments on one chip. In the inter-assay, we analyzed the same serum sample twice each day for ten consecutive days. In addition, we determine AFP in thirty clinical human serum samples. We compare the results with the values obtained by electrochemiluminescence (ECL) immunoassay of the Roche Elecsys AFP kit and Cobas e411 automated immunoassay chemistry analyzer (Basel, Switzerland) which are commonly used in hospitals. All the experiments using clinical human serum samples are approved by the Ethic Committee of the 306th Hospital of PLA (2015-04). RESULTS AND DISCUSSION The Changes of Hydrophilicity of Polymeric Surfaces Caused by Modification. The effectiveness of hydrophilic surfaces by the one-step modification is assessed by measuring static water droplet contact angles on the modified surfaces which are compared with unmodified surfaces. We observe that the contact angles became smaller after modification and the surfaces change to be hydrophilic. Figure 1A shows the images of water droplets on the surface of modified and unmodified polymeric slabs and their contact angles. By modification, the contact angle of water droplets on the COC slab surface changes from 75.5° to 33.0°. For PC, the water contact angle of changes from 73.9° to 28.3°. For PMMA, the contact angle changes from 71.4° to 17.6°. For PS, the contact angle changes from 84.2° to 31.4°. The amounts of changes arrange from 42.5° to 53.8°, and PMMA slab surface gains the largest change. The results show that the contact angles on these four kinds of polymeric surfaces change obviously and the surfaces have changed to be hydrophilic through the one-step modification. Due to poly-norepinephrine bitartrate has hydroxyl groups on the macromolecular chains, its modification improves the hydrophilicity of the polymeric surfaces. Norepinephrine, a kind of catecholamine, has been reported for surface modification,31, 32 but its toxicity is high. As the toxicity of norepinephrine bitartrate is much less than norepinephrine according to the Material Safety Data Sheet (MSDS), we choose norepinephrine bitartrate as the modification reagent. We have tested norepinephrine bitartrate with different concentrations for modification, and 20 mg mL-1 is the most appropriate concentration according to our tests. We monitor the contact angles of the modified COC, PC, PMMA, and PS slabs during consecutive ten weeks (Figure 1B). Although we are not clear about the mechanism of the modification, the results show that the modification is chemically stable over time. The water contact angle of modified COC slab remains about 35.1° within ±1.7° for 10 weeks (Figure 1B). For PC, the contact angle remains about 31.1° within ±1.6°. For PMMA, the contact angle remains about 18.8° within ±1.3°. For PS, the contact angle remains about 33.4° within ±1.9°. As the contact angle of the modified PMMA slab is the smallest, we choose it for further study. Auto-injection Performance in Modified Channels and Flow Characteristics. Auto-injection is very important in the applications of chips. High performance of auto-injection can avoid using pumps or other dynamic systems and makes the whole device simple and easy to operate. To observe the characteristics of liquid flow in the channels, we introduce a 10 µL droplet with red dye upon the inlet of the modified PMMA microfluidic chip (Figure 2A). The PMMA channel is 300 µm in width, 300 µm in height and 100 mm in length. The drop flows into the inlet and passes through the channel to reach the

Figure 1. The changes of contact angles of polymeric surfaces caused by modification. (A) The contact angles of cycloolefin copolymeric (COC), polycarbonate (PC), polymethylmethacrylate (PMMA), and polystyrene (PS) slabs surfaces before and after the modification. (B) The contact angle changes of the modified COC, PC, PMMA, and PS slabs during consecutive ten weeks.

outlet in 16 seconds without any external forces. In the first 2 seconds, the liquid moves relatively fast and covers almost half of the total channel. In the next 14 seconds, its moving speed slows down until it reaches the outlet. This presumably because that the resistance became larger as the drop has more contact areas and resistance with the inner walls of the channel and the bottom PDMS sheet when the drop flows into the inlet and passes through the channel. In the control test, we also introduce a droplet of red-colored dye upon the inlet of the channel which is unmodified, and the droplet stays at the inlet without moving. The Video S1 shows in supporting information. To test the long-term performance of auto-injection after surface modification, a batch of chips which were modified 2.5 years ago are investigated for the auto-injection. The droplets can also flow into the inlet and pass through the modified channels without any external forces. The velocity is the same as the newly modified ones (Figure 2B). It confirms that the modification is extremely durable. Three samples are tested to calculate the velocity of the drops flow through the newly modified channel and channel modified 2.5 years ago respectively. The error bars are calculated by standard deviations. To explore the effect of the widths of the channels on the speeds of flow in the modified channels, we test channels with the same height and the same length but different widths. We introduce a 20 µL droplet with red dye upon the inlet of the modified PMMA microfluidic chip and the drop flows into the inlet and passes through the channel to reach the outlet. We can see from Figure 2C that the droplet flows faster in the wider channels, and the flow velocity of the drop in the channel with the width of 700 µm is about four times as that in the channel with the width of 300 µm. These observations are presumably so because the resistance in wide channels is lower than in that the narrow ones. To explore whether the gravity may have an effect on the rate of flow, we test channels with the same width, height and length, but the thicknesses of top PMMA fluidic layers are different. These channels have different thickness of fluids in their inlet reservoirs. We found the flow velocities of the droplets in these channels are not significantly different, and the



times they take the drops to flow from the inlet to the outlet are almost the same, within 4 seconds (Figure 2D). Thus, we consider the effect of the gravity does not dominate in such conditions.

Figure 2. Auto-injection performance in modified channels and flow characteristics. (A) Auto-injection performance in the PMMA channel of 300 µm × 300 µm × 100 mm. In the control test, the droplet stays at the inlet channel which is unmodified. The droplet with red dye flows through the modified channel to reach the outlet within 16 seconds. (B) The velocities of the drops flow through the newly modified channel and channel modified 2.5 years ago. (C) The times they take the drops to flow from the inlet to the outlet in the PMMA channels with the same height (300 µm) and the same length (100 mm) but different widths (300, 400, 500, 600, and 700 µm), and the height of the top PMMA fluidic layer is same (2 mm). (D)The times they take the drop to flow from the inlet to the outlet in the PMMA channels with 600 µm in width, 300 µm in height, 100 mm in length but the thicknesses of the top PMMA fluidic layers are different (2, 3, 4, 5, and 6 mm).

Reduction of Background Noise in Immunoassays on Modified Chips. To investigate the effect of the modification on the chips for application performance, we carry out chemiluminescence immunoassays based on microfluidic chips for AFP at three different concentration levels. The results of immunoassay using modified PMMA chips and unmodifed chips are compared in Figure 3. We note that the background noise where PMMA channels are unmodified is obviously higher than that modified (Figure 3A). Also, the enlarged gray value analysis in the red dashed frame area of Figure 3A is presented in Figure 3B. The gray value of peak background noise where unmodified PMMA channel placed is about 76, while the value of peak background noise where modified PMMA channel placed is about 64. The SNRs of these three concentration levels using unmodified channels are arrange from 10.3 to 48.6. While the SNRs using modified channels are arrange from 104.3 to 313.1 (Figure 3C). And the SNRs of these three concentration levels are enhanced about 10, 13 and 6 times, respectively. We use ImageJ to calculate the gray intensities of the square where the antibody-antigen-antibody sandwich complex is formed as signals and the square with equal area just below as noises. The diagram of this process is shown in Figure S2. Thus, we can obviously enhance the chip performance using this one-step modification method. The improvement of SNR in this approach most likely comes from enhancement in the efficiency in cleaning the inner walls of the channels. From Figure 3A, we can see that background noise at the junctures of the bottom PDMS sub-

strate and the vertical inner walls of unmodified channels is obviously higher than where modified PMMA channel placed. Maybe this because the hydrophobic characteristics of the surface of inner wall in unmodified channel prevent the cleaning buffer from reaching into the small gaps at the junctions, and it is relatively inefficient to clean the fouling at the junctures. As we can see from the result of immunoassay on unmodified chips in Figure 3A and 3B, the peaks of the gray value are all appear at these junctures. However, in this assay, the background noise where using modified channels can be significantly decreased because the cleaning buffer can flow into the small gaps at the junctions and clean away the fouling there. Thus, the effect of cleaning in the modified microfluidic channels is much better than that in the unmodified channels, which result in low background noise and high SNR.

Figure 3. Chemiluminescence immunoassay for alphafetoprotein (AFP) on modified and unmodified PMMA microfluidic chips. (A) Different concentrations of AFP standard solution (1 ng ml-1, 3 ng ml-1, 5 ng ml-1) are detected in unmodified and modified channels. The gray spots on the chip demonstrated the immunoassay signals for AFP. (B) The gray value analysis of the red dashed frame area in A; this area indicates the non-specific signals. The X axis shows the horizontal distance in the red dashed frame area, and the Y axis shows the gray value of the pixels. (C) The signal-to-noise ratios (SNR) of using unmodified channels and modified channels.

Reproducibility and Accuracy of Clinical Serum Sam-ples Assays. To evaluate the reliability and application potential, we determine the reproducibility and the accuracy of immunoassay for clinical human serum samples using modified channels. By using the newly modified channels, the intraassay CV determined through 49 parallel measurements on one chip of the same serum sample is 5.63 %, and the interassay CV determined through twice each day for 10 consecutive days of the same serum sample is 7.21 % (Figure 4A). By using the channels modified 2.5 years ago, the intra-assay CV of the same serum sample is 5.77 %, and the inter-assay CV of the same serum sample is 7.63 %. By contrast, in the control tests which use unmodified channels, the intra-assay CV is 9.87 % and the inter-assay CV is 11.3 %. We analyze the coefficient of variation between unmodified channels, newly modified channels, and channels modified 2.5 years ago in the intra-assays and the inter- assays by one-way analysis of variance via SPSS statistical software (IBM, USA). There is significant difference between modified channels (newly modified or modified 2.5 years ago) and control group (unmodified


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Analytical Chemistry channels). The background noise caused by nonspecific adsorption is significant and reduces the reproducibility in analysis. After modification, the background noise caused by nonspecific adsorption is reduced and the background noise is low and uniform, which increases the reproducibility. The intraassay CV and the inter-assay CV of immunoassay which carried in the newly modified channels are similar with those in the channels modified 2.5 years ago, which further confirms that the modification is extremely stable. We also determine AFP in thirty clinical human serum samples, and compare the results with the values obtained by electrochemiluminescence (ECL) immunoassay of the Roche Elecsys AFP kit on a Cobas e411 automated immunoassay chemistry analyzer. As can be seen in Figure 4B, the AFP concentrations of thirty serum samples measured in modified chips strongly correlated with the values obtained using Roche's ECL method. The best fit regression equation is Y = 2.087 + 0.936X, and the coefficient is 0.9827, which shows the accuracy of this method is reliable. The detection limitation calculated as three time of signal-to-noise ratio was 0.09 ng mL-1 for AFP by detecting a diluted human serum sample. In the immunoassays, we have not found the samples could be contaminated by the modification.

serum samples, the results are strongly correlated with the values obtained using commercial products. Thus, we believe that this straightforward and ultrastable modification method can improved the performance of microfluidic chips in immunoassays, and can be used in other assays in analytical technology and biosensing for further research.

ASSOCIATED CONTENT Supporting Information Figure S1. Schematic diagram of microfluidic chips. (PDF) Figure S2. Schematic diagram of calculating signal-to-noise ratios. (PDF) Video S1. Auto-injection performance in the modified channel (down) and control test in the unmodified channel (up).(WMV)

AUTHOR INFORMATION Corresponding Author * Corresponding authors: [email protected] (Xingyu Jiang), [email protected] (Feng Qu), [email protected] (Yong Xia).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the Ministry of Science and Technology of China (2013YQ190467), the National Science Foundation of China (21675012, 81361140345, 51373043, 21535001), and Chinese Academy of Sciences (XDA09030305) for financial support. Figure 4. Reproducibility and accuracy of clinical serum samples assays. (A) The reproducibility of immunoassay (coefficient of variation) for clinical human serum samples using unmodified channels, newly modified channels, and channels modified 2.5 years ago. Asterisk indicates significant difference between modified channels and control group (unmodified channels) (**p < 0.01). (B) Correlation between Roche ECL immunoassay and the microfluidic immunoassay for the detection of AFP in thirty clinical human serum samples.

CONCLUSIONS In this study, we develop straightforward and ultrastable surface modification method for polymeric surface using norepinephrine bitartrate. We modify polymeric surfaces with norepinephrine bitartrate in one step and the hydrophilicity of the polymeric surface is improved dramatically. Autoinjection, which is big challenge for most polymeric chips, can be performed in the modified channels even in the channels modified 2.5 years ago, confirming that the modified surfaces are extremely stable and the period is more than twice as reported before24. In the immunoassays for AFP, we find that the modification could reduce the background noise caused by nonspecific adsorption and enhance the SNR significantly. In a lot of bioanalytical experiments, nonspecific adsorption perplexes researchers to get clear pictures or results. Thus, this method may be very helpful for them to reduce the background noise. We also find the intra-assay CV and inter-assay CV of immunoassays are reduced dramatically and have hardly changed in at least 2.5 years, which shows this method can improve the reproducibility of immunoassay significantly and ultrastably. In the detection for AFP in thirty clinical human

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TOC Auto-injection performance in the modified channel.


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Straightforward and Ultrastable Surface Modification of Microfluidic

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