- Email: [email protected]
Development of Integrated Microfluidic Magnetic Bioprocessor for Multi-biomarker Detection
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 41, Issue 9, September 2013 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2013, 41(9), 1302–1307.
RESEARCH PAPER
Development of Integrated Microfluidic Magnetic Bioprocessor for Multi-biomarker Detection LIAN Jie1,2, ZHOU Wen-Wen1,3, SHI Xi-Zeng1,4, GAO Yun-Hua1,* 1
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2 College of Forensic Science, People’s Public Security University of China, Beijing 100038, China 3 Avantes China Co., Ltd., Beijing 100102, China 4 Dongguan Bosh Biotechnologies Ltd., Dongguan 523808, China
Abstract: By integrated magnetic sensors and microfluidic system, a microfluidic magnetic bioprocessor was developed for the rapid detection of multi-target biomarkers. Three digestive system tumor makers, -fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) were simultaneously detected in immune reactions in the microchannel as a model system to evaluate this multi-target detection system performance. The experiment parameters including injecting flow velocity, immune reaction time and rinsing rate for each step were optimized. The standard curves were established for three tumor markers detection in serum samples, with the detection limits of 0.1 ng mL–1 (CEA), 0.1 ng mL–1 (AFP), 30 U mL–1 (CA19-9) and the dynamic ranges spanning four orders of magnitude. Multi-biomarker analysis could be completed within 30 min, and good linear relations were obtained between the results of magnetic bioprocessor and ELISA method, otherwise, the former had the advantages such as fast detection and high sensitivity. Key Words: Microfluidic system; Magnetic tunnel junction biosensor; Multi-biomarker detection
1
Introduction
In recent years, microfluidic technique has been rapidly developed[1,2] , and caused extensive concern in the chemical, pharmaceutical and life science researches, becoming a research focus at home and abroad. Microfluidic chip, also known as "Lab on a chip"[3], is an integration technology that integrates and miniaturizes the separating, reaction and mixing devices in general laboratories onto a chip on the basis of microelectromechanical technology. The biochemical reaction or analysis can be realized on a chip through the complexed and precise operation of microfluid, resulting in wide applications in the field of biological analysis, with the advantages of small size, portable device, less sample size,
reaction speed , parallel processing, and so on[4–9]. Biosensor, as an important branch of biological analysis technique, has combined related the fields of science such as life science, analytical chemistry, physics, computer science and information technologies. The biosensor technique can quarantine the analyte in a short time and has been applied in the fields of medical testing, environmental monitoring, food security, military security[10–12]. Among various kinds of biosensors, magnetic tunnel junction (MTJ) sensor is a novel type of biological sensors utilizing giant magnetoresistance effect, with super paramagnetic particles as the detection probe[12–16]. MTJ sensor could be expected as a portable, fast and new technical platform for biology[10–14], pharmaceutical research[15], and medical testing[16], with a series of new
Received 15 March 2013; accepted 1 May 2013 * Corresponding author. Email: [email protected] This work was supported by the National Key Technology R&D Program of China (No.2013BAI03B), the Knowledge Innovation Program of the Chinese Academy of Sciences Grant (No. KJCX2-YW-M15), and the Comprehensive Strategic Cooperation Program between Guangdong Province and Chinese Academy of Science (No. 2011A090100036). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60681-7
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
features such as small size, low cost, low background, high sensitivity and high throughput. Tumor markers are some biochemical materials in body fluids or cells, indicating the patients suffering from cancer. Also, the combined use of multiple tumor markers would improve the overall accuracy to discern the healthy from patients, especially in early stage of cancer, widely used in clinical early detection, diagnosis and treatment of different kinds of cancers[17–20]. The common detection methods of tumor markers mainly include enzyme-linked immunoassay (ELISA), radioimmunoassay, fluorescence immunoassay, and gold immunochromatography assay, which are designed for single target (tumor marker) per test with long time-consuming and complicated operation. By combining with the technique of microfluidic system, it can improve the automation of the tumor markers detection, and can easily undertake a quick multi-target test of high sensitivity and high throughput with MTJ sensors, providing an efficient detection method for simultaneous detection of tumor markers. In this study, an integrated microfluidic magnetic bioprocessor-on-chip was designed and developed, and the feasibility was evaluated through the simultaneous and quantitative detection of three digestive system tumor markers.
2 2.1
Experimental Instrumentation and chemicals
As shown in Fig.1, the prototype of the magnetic biosensor tester was jointly developed by Technical Institute of Physics and Chemistry of Chinese Academy of Science and Dongguan Bosh Biotechnologies Ltd., China. The dimension parameters of MTJ sensor array chip[16] were 6.6 mm× 10.7 mm × 0.75 mm (length × width × thickness), produced by Magic Technologies, US. Vacuum Drying Oven DZF-6020 was purchased from Shanghai Jing Hong Laboratory Instrument Co., Ltd. All the liquid movements were driven by the syringe micro-pump (L0107-1, Baoding Longer Precision Pump Co., Ltd.). The results of static reaction were observed through fluorescence microscope (BX51, Olympus Optical). Ultrapure water (18.3 M cm ) was obtained from EASY pure RF water system (Barnstead International, United States) and used throughout. The antigens of -Fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and the related antibodies and biotin labeled antibodies were purchased from Fitzgerald Industries International, United States. Bovine serum albumin (BSA) and pure fetal bovine serum (excellent level) were obtained from Beijing Xinjingke Biotechnology, China. Avidin-superparamagnetic particles (Avidin-beads) were supplied by Ademtech, France. -Glycidoxypropyl trimethoxysilane (GPTS) were purchased
from Alfa Aesa (more than 97% purity). Sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate and sodium bicarbonate were purchased from Merck, USA). 2.2
Immune principle of magnetic bioprocessor
Magnetic bioprocessor accomplishes the analysis of biological molecules with superparamagnetic particles as probes, based on the response of MTJ sensor array chip to the probes captured by those biomolecules. An external magnetic field is used to magnetize the superparamagnetic particles to generate a magnetic field (Fig.2). The magnetic field generated disturbs under the MTJ sensor to produce a resistivity change. And then the resistivity change was transformed into a current signal, and the number of magnetic particles on the chip was obtained further[13]. The principle of tumor markers detection is sandwich immune response: the first antibody on the surface of the chip (1st Ab), antigen in the blood, and the second antibody modified by biotin (2nd Ab-Biotin) were combined together to form a sandwich structure through the specificity of immune response. Superparamagnetic particles (Avidin-Bead) were captured onto the chip surface through 2nd Ab-Biotin by the affinity of avidin and biotin. The MTJ array was then measured again to obtain the information of Superparamagnetic particles which remain on the chip due to specific immuno-reaction processes. Thus, the quantitative detection of target molecules in the analyte was achieved by the above procedure. A
B
Fig.1 Photographs of tester (A) and test cartridge (B) of MTJ array biosensor (40 mm × 50 mm)
Fig.2 Schematic of immune reactions on MTJ sensor
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
2.3
Design of microfluidics test card in magnetic bioprocessor
The programmed reaction, separation and test of the analyte online were carried out by the microfluidic test cartridge integrated magnetic sensors modified with specific antibodies. As shown in Fig.3A, the test card was a three-layer structure, made of cartridge, PDMS layer with microchannel layer and Printed Circuit Board with MTJ chip (PCB with MTJ chip). There were four inlets on the cartridge part for the injection of sample, rinsing buffer, 2nd Ab-Biotin, and Avidin-Bead, respectively. The microchannels in the PDMS layer were designed, and the liquid was injected from different inlets under the drive of the syringe micropump, flowing through different zones of the chips into the waste pool (Fig.3B). The simultaneous detection of multiple targets was achieved by the means of immobilizing different 1st antibodies onto different regions of a single chip. 2.4 2.4.1
Experimental procedure
2.4.2
Immune reaction and magnetic detection
The analysis process was performed at room temperature. 1st Ab were immobilized onto the selective zones of GPTStreated MTJ chips by dropping 500 nL of corresponding coating solutions[21], which were 1st Ab anti-AFP, 1st Ab anti-CEA, 1st Ab anti-CA19-9, or BSA with the concentration of 20 mg L–1 in coating buffer, respectively. After the chips were incubated for 10 min under 90% relative humidity, 1% BSA-PBST was added onto the chip to block the zones of the chip surface. The chips were assembled into test card after 5 min. A 40-L of the analyte and a 40-L of 2nd Ab-Biotin mixture were injected and kept static reaction at room temperature for certain time, and an 80-L of rinsing buffer was injected to remove unreacted regents. Then a 40-L of Avidin-Beads was injected with certain reaction time, and following an 80-L of rinsing buffer was injected to remove unbounded particles. The number of magnetic particles was reported after loading test card into the tester, and the correlation between the number of magnetic particles and the concentration of antigens in the analyte was obtained.
Buffers and diluents
3 The sodium carbonate buffer (pH 9.6), as the coating buffer, was prepared by dissolving 0.159 g of Na2CO3 and 0.293 g of NaHCO3 in 100 mL of ultrapure water. Phosphate buffer solution (PBS, pH 7.4, adjusted by phosphoric acid and NaOH solution) was prepared by dissolving 1.439 g of KH2PO4 and 1.439 g of Na2HPO4 in 800 mL ultrapure water. PBST was prepared by adding 100 mL Tween 20 into PBS. 0.1% BSA-PBST was prepared by adding 0.1 g BSA into 100 mL PBST solution and mixing well, as a cleaning fluid, the diluents of 2nd Ab-Biotin and Avidin-Beads. 1% BSA-PBST was prepared by adding 0.1 g BSA into 100 mL PBST solution and mixing well, as the blocking buffer. The samples were prepared by diluting a certain amount of antigens diluted with fetal bovine serum. All the above solutions were added NaN3 as anti-microbial agents at a final concentration of 0.04% (w/V), and stored at 4 ºC until use.
Avidin-Beads 2nd Ab-Biotin
3.1
Results and discussion Optimization of system parameters
The combination of antigen and antibodies depends on the complementarity between the two kinds of molecular structure and affinity in immunoreactions, and the required reaction time is influenced by a variety of factors and reaction conditions, meanwhile the efficiency of the immune response in microfluidic system is highly influenced by flow rate in microchannels and the surface tension on the chip. In the experiment, the immune reactions and rinsing processes were realized by the means of liquid flowing in microfluidic channels driven by syringe micropump. The f analysis conditions of injection rates and immune reaction times of reaction reagents, as well as rinsing conditions on the surface of the chip were optimized. Due to the volumes of the cartridge
Rinsing Buffer Sample
Waste Cartridge
AvidinBeads
BSA (negative control) Anti-AFP Anti-CEA Anti-CA19-9
2nd AbBiotin Rinsing Buffer
Chip
PDMS Layer Microchannel above the chip
PCB with MTJ chip
Sample Waste
Fig.3 Design sketch of MTJ Biosensor test card (A) and the fluidic channel (B)
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
chambers were fixed, the injection speed and rinsing conditions were determined by measuring the reagent injection times and rinsing buffer injection time. The experiments were designed respectively for three kinds of digestive system tumor markers, and only the optimization of the system parameters for CEA analysis was listed below. First, the concentrations of 2nd Ab-Biotin and Avidin-Beads were discussed when moderate microfluidic conditions and sufficient reaction time (15 min per step) were set, and the optimized concentrations of 2nd Ab-Biotin and Avidin-Beads were respectively 20 g mL–1 and 1 mg mL–1 (diluted by 10 times). After the volume of the rinsing buffer was set as 80 L, a series of injection times of rinsing buffer were employed to study the impact of the rinsing speed on specific reaction (with the concentrations of CEA of 1 ng mL–1) and nonspecific adsorption (without CEA) of antigen and antibody. As shown in Fig.4A, the number of nonspecific adsorbed magnetic particles decreased slightly with the shortening rinsing time, and the same trend was observed for the number of specific reacted magnetic particles. The optimal rinsing conditions for injection was to inject 80 L rinsing buffer in 60 s, by which a high signal to noise ratio was obtained. Immune response on the surface of the chip was shown in Fig.2. The interaction between antigens in the serum and antibodies on the chip was completed during the first incubation process in the cartridge, and the reaction efficiency of this step directly affected the formation of the final “Ab-Antigen-Ab” sandwich structure. Thus, the incubation time of the analyte with antigens had a direct and important influence on the immune analysis of tumor markers. Different static incubation times of analyte containing CEA were investigated under the concentration of 1 ng mL–1 CEA. The number of magnetic particles in the detected region increased
with the increase of incubation reaction time, and the increase in extent decreases after 10 min, roughly reaching a balance (Fig.4B). The static reaction times of 2nd Ab-Biotin and Avidin-Beads were also investigated. The reaction reached a general saturated status within 5 min. Therefore, the optimized reaction time for the three-step immune responses in the immune analysis of CEA was 10, 5 and 5 min, respectively. In microfluid bioprocessor, the system parameters were investigated for the analysis of AFP, CEA and CA19-9 respectively. The optimal reaction time of the three kinds of tumor markers were almost the same, while the optimal rinsing conditions were no difference (Table 1), so 60 s was identified as the optimal time in multi-target joint detection. Therefore, it could accomplish the detections of the above three tumor markers at the same time within 30 min by using the bioprocessor. 3.2
Joint detection of multiple digestive system tumor markers in magnetic bioprocessor
Based on the optimization of system parameters, different kinds of 1st Ab were coated on different regions of the chip, and the joint detections of three kinds of digestive system tumor markers AFP, CEA and CA19-9 were carried out. The standard working curves are shown in Fig.5. The Multi-target detection of a sample of three digestive system tumor markers could be finished within 30 min by the magnetic bioprocessor with the detection limits of 0.1 ng mL–1 for AFP, 0.1 ng mL–1 for CEA and 30 U mL–1 for CA19-9. The linear ranges of detection spanned four orders of magnitude, completely satisfying the requirements of clinical diagnostic. As shown in Fig.5, the standard working curves of three tumor markers were different in the low concentration
Fig.4 Optimization of rinsing time (A) and static reaction time (B) of CEA Table 1 Optimization of rinsing conditions in PDMS microchannels Reaction time of Antigen (min)
Optimized rinsing time of AFP (s)
Optimized rinsing time of CEA (s)
Optimized rinsing time of CA19-9 (s)
5
80
60
60
10
40
60
60
15
40
40
40
Optimized rinsing time of multi-targets (s) 60
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
Fig.5
Standard curves for tumor markers by MTJ biosensor: detection of low-concentration (A. AFP, C.CEA, E. CA19-9) and high-concentration (B. AFP, D.CEA, F. CA19-9)
ranges and high concentration ranges, and the working curves all had good linearity (correlation coefficients close to 1.0). The deviations of analyzing samples of CEA are summarized in Table 2. The deviation was 11% at the concentration of 0.1 ng mL–1, 7.6% at the concentration of 1 ng mL–1, and as the concentration was above 1 ng mL–1, the deviation was less than 10%, meeting the clinical requirement for detection precision. Clinical serum samples were analyzed by the magnetic bioprocessor and the clinical used commercial ELISA kits. Correlation analysis was run between the detection results of two methods (Fig.6), and the correlation coefficients of two methods were 0.947 (CEA), 0.995 (AFP), 0.947 (CA19-9), respectively. The detection results of simultaneous analysis of three tumor markers by the magnetic bioprocessor agreed with ELISA results. By using the magnetic bioprocessor for the multi-biomarker test, the detection range and detection limit all reached the level of clinical use, with high sensitivity and rapid detection time of 30 min, which was much shorter than that of the conventional ELISA method (2–4 h per single target test). The optimal microfluidic reactions realized a series of biomarkers immune analysis; greatly reduced the time needed for the test process, and also realized the joint multi-target detection on the magnetic sensitive biosensors.
Fig.6 Correlation between three tumor markers in serum measured by MTJ biosensor and ELISA kit
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R, Rife J C, Stanton M W, Whitman L J. Biosensors and Bioelectronics, 2007, 23(2): 191–200 [15] Gaster R S, Hall D A, Wang S X. Lab Chip, 2011, 11(5): 950–956 [16] Lian J, Chen S, Qiu Y, ZhangS, ShiX, Gao Y. Journal of Applied Physics, 2012, 111: 07B315
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[11] Sun Y, Bai Y, Song D, Li X, Wang L, Zhang H. Biosens. Bioelectron., 2007, 23(4): 473–478 [12] Schotter J, Kamp P B, Becker A, Pühler A, Reiss G, Brückl H. Biosensors and Bioelectronics, 2004, 19(10): 1149–1156 [13] Shen W, Schrag B D, Carter M J, Xie J, Xu C, Sun S, Xiao G. J.Appl. Phys., 2008, 103(7): 07A306 [14] Mulvaney S P, Cole C L, Kniller M D, Malito M, Tamanaha C
Ohkura H, Kodaira S, Okajima K, Nakazato H, Tumor Marker C. Gastric Cancer, 2003, 6(3): 142–145 [19] Kayaba H, Nihon Igaku Hoshasen Gakkai zasshi. Nippon Acta Radiologica, 2003, 63(4): 133–139 [20] Flejou J F. Annales de Pathologie, 2011, 31(5 Suppl): S27–S31 [21] Zhou W W, Lian J, Hu K J, Gao Y H, Xu B. Acta Phys. Chim. Sin., 2010, 26(10): 2821–2827
Cite this article as: Chin J Anal Chem, 2013, 41(9), 1302–1307.
RESEARCH PAPER
Development of Integrated Microfluidic Magnetic Bioprocessor for Multi-biomarker Detection LIAN Jie1,2, ZHOU Wen-Wen1,3, SHI Xi-Zeng1,4, GAO Yun-Hua1,* 1
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2 College of Forensic Science, People’s Public Security University of China, Beijing 100038, China 3 Avantes China Co., Ltd., Beijing 100102, China 4 Dongguan Bosh Biotechnologies Ltd., Dongguan 523808, China
Abstract: By integrated magnetic sensors and microfluidic system, a microfluidic magnetic bioprocessor was developed for the rapid detection of multi-target biomarkers. Three digestive system tumor makers, -fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) were simultaneously detected in immune reactions in the microchannel as a model system to evaluate this multi-target detection system performance. The experiment parameters including injecting flow velocity, immune reaction time and rinsing rate for each step were optimized. The standard curves were established for three tumor markers detection in serum samples, with the detection limits of 0.1 ng mL–1 (CEA), 0.1 ng mL–1 (AFP), 30 U mL–1 (CA19-9) and the dynamic ranges spanning four orders of magnitude. Multi-biomarker analysis could be completed within 30 min, and good linear relations were obtained between the results of magnetic bioprocessor and ELISA method, otherwise, the former had the advantages such as fast detection and high sensitivity. Key Words: Microfluidic system; Magnetic tunnel junction biosensor; Multi-biomarker detection
1
Introduction
In recent years, microfluidic technique has been rapidly developed[1,2] , and caused extensive concern in the chemical, pharmaceutical and life science researches, becoming a research focus at home and abroad. Microfluidic chip, also known as "Lab on a chip"[3], is an integration technology that integrates and miniaturizes the separating, reaction and mixing devices in general laboratories onto a chip on the basis of microelectromechanical technology. The biochemical reaction or analysis can be realized on a chip through the complexed and precise operation of microfluid, resulting in wide applications in the field of biological analysis, with the advantages of small size, portable device, less sample size,
reaction speed , parallel processing, and so on[4–9]. Biosensor, as an important branch of biological analysis technique, has combined related the fields of science such as life science, analytical chemistry, physics, computer science and information technologies. The biosensor technique can quarantine the analyte in a short time and has been applied in the fields of medical testing, environmental monitoring, food security, military security[10–12]. Among various kinds of biosensors, magnetic tunnel junction (MTJ) sensor is a novel type of biological sensors utilizing giant magnetoresistance effect, with super paramagnetic particles as the detection probe[12–16]. MTJ sensor could be expected as a portable, fast and new technical platform for biology[10–14], pharmaceutical research[15], and medical testing[16], with a series of new
Received 15 March 2013; accepted 1 May 2013 * Corresponding author. Email: [email protected] This work was supported by the National Key Technology R&D Program of China (No.2013BAI03B), the Knowledge Innovation Program of the Chinese Academy of Sciences Grant (No. KJCX2-YW-M15), and the Comprehensive Strategic Cooperation Program between Guangdong Province and Chinese Academy of Science (No. 2011A090100036). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60681-7
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
features such as small size, low cost, low background, high sensitivity and high throughput. Tumor markers are some biochemical materials in body fluids or cells, indicating the patients suffering from cancer. Also, the combined use of multiple tumor markers would improve the overall accuracy to discern the healthy from patients, especially in early stage of cancer, widely used in clinical early detection, diagnosis and treatment of different kinds of cancers[17–20]. The common detection methods of tumor markers mainly include enzyme-linked immunoassay (ELISA), radioimmunoassay, fluorescence immunoassay, and gold immunochromatography assay, which are designed for single target (tumor marker) per test with long time-consuming and complicated operation. By combining with the technique of microfluidic system, it can improve the automation of the tumor markers detection, and can easily undertake a quick multi-target test of high sensitivity and high throughput with MTJ sensors, providing an efficient detection method for simultaneous detection of tumor markers. In this study, an integrated microfluidic magnetic bioprocessor-on-chip was designed and developed, and the feasibility was evaluated through the simultaneous and quantitative detection of three digestive system tumor markers.
2 2.1
Experimental Instrumentation and chemicals
As shown in Fig.1, the prototype of the magnetic biosensor tester was jointly developed by Technical Institute of Physics and Chemistry of Chinese Academy of Science and Dongguan Bosh Biotechnologies Ltd., China. The dimension parameters of MTJ sensor array chip[16] were 6.6 mm× 10.7 mm × 0.75 mm (length × width × thickness), produced by Magic Technologies, US. Vacuum Drying Oven DZF-6020 was purchased from Shanghai Jing Hong Laboratory Instrument Co., Ltd. All the liquid movements were driven by the syringe micro-pump (L0107-1, Baoding Longer Precision Pump Co., Ltd.). The results of static reaction were observed through fluorescence microscope (BX51, Olympus Optical). Ultrapure water (18.3 M cm ) was obtained from EASY pure RF water system (Barnstead International, United States) and used throughout. The antigens of -Fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and the related antibodies and biotin labeled antibodies were purchased from Fitzgerald Industries International, United States. Bovine serum albumin (BSA) and pure fetal bovine serum (excellent level) were obtained from Beijing Xinjingke Biotechnology, China. Avidin-superparamagnetic particles (Avidin-beads) were supplied by Ademtech, France. -Glycidoxypropyl trimethoxysilane (GPTS) were purchased
from Alfa Aesa (more than 97% purity). Sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate and sodium bicarbonate were purchased from Merck, USA). 2.2
Immune principle of magnetic bioprocessor
Magnetic bioprocessor accomplishes the analysis of biological molecules with superparamagnetic particles as probes, based on the response of MTJ sensor array chip to the probes captured by those biomolecules. An external magnetic field is used to magnetize the superparamagnetic particles to generate a magnetic field (Fig.2). The magnetic field generated disturbs under the MTJ sensor to produce a resistivity change. And then the resistivity change was transformed into a current signal, and the number of magnetic particles on the chip was obtained further[13]. The principle of tumor markers detection is sandwich immune response: the first antibody on the surface of the chip (1st Ab), antigen in the blood, and the second antibody modified by biotin (2nd Ab-Biotin) were combined together to form a sandwich structure through the specificity of immune response. Superparamagnetic particles (Avidin-Bead) were captured onto the chip surface through 2nd Ab-Biotin by the affinity of avidin and biotin. The MTJ array was then measured again to obtain the information of Superparamagnetic particles which remain on the chip due to specific immuno-reaction processes. Thus, the quantitative detection of target molecules in the analyte was achieved by the above procedure. A
B
Fig.1 Photographs of tester (A) and test cartridge (B) of MTJ array biosensor (40 mm × 50 mm)
Fig.2 Schematic of immune reactions on MTJ sensor
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
2.3
Design of microfluidics test card in magnetic bioprocessor
The programmed reaction, separation and test of the analyte online were carried out by the microfluidic test cartridge integrated magnetic sensors modified with specific antibodies. As shown in Fig.3A, the test card was a three-layer structure, made of cartridge, PDMS layer with microchannel layer and Printed Circuit Board with MTJ chip (PCB with MTJ chip). There were four inlets on the cartridge part for the injection of sample, rinsing buffer, 2nd Ab-Biotin, and Avidin-Bead, respectively. The microchannels in the PDMS layer were designed, and the liquid was injected from different inlets under the drive of the syringe micropump, flowing through different zones of the chips into the waste pool (Fig.3B). The simultaneous detection of multiple targets was achieved by the means of immobilizing different 1st antibodies onto different regions of a single chip. 2.4 2.4.1
Experimental procedure
2.4.2
Immune reaction and magnetic detection
The analysis process was performed at room temperature. 1st Ab were immobilized onto the selective zones of GPTStreated MTJ chips by dropping 500 nL of corresponding coating solutions[21], which were 1st Ab anti-AFP, 1st Ab anti-CEA, 1st Ab anti-CA19-9, or BSA with the concentration of 20 mg L–1 in coating buffer, respectively. After the chips were incubated for 10 min under 90% relative humidity, 1% BSA-PBST was added onto the chip to block the zones of the chip surface. The chips were assembled into test card after 5 min. A 40-L of the analyte and a 40-L of 2nd Ab-Biotin mixture were injected and kept static reaction at room temperature for certain time, and an 80-L of rinsing buffer was injected to remove unreacted regents. Then a 40-L of Avidin-Beads was injected with certain reaction time, and following an 80-L of rinsing buffer was injected to remove unbounded particles. The number of magnetic particles was reported after loading test card into the tester, and the correlation between the number of magnetic particles and the concentration of antigens in the analyte was obtained.
Buffers and diluents
3 The sodium carbonate buffer (pH 9.6), as the coating buffer, was prepared by dissolving 0.159 g of Na2CO3 and 0.293 g of NaHCO3 in 100 mL of ultrapure water. Phosphate buffer solution (PBS, pH 7.4, adjusted by phosphoric acid and NaOH solution) was prepared by dissolving 1.439 g of KH2PO4 and 1.439 g of Na2HPO4 in 800 mL ultrapure water. PBST was prepared by adding 100 mL Tween 20 into PBS. 0.1% BSA-PBST was prepared by adding 0.1 g BSA into 100 mL PBST solution and mixing well, as a cleaning fluid, the diluents of 2nd Ab-Biotin and Avidin-Beads. 1% BSA-PBST was prepared by adding 0.1 g BSA into 100 mL PBST solution and mixing well, as the blocking buffer. The samples were prepared by diluting a certain amount of antigens diluted with fetal bovine serum. All the above solutions were added NaN3 as anti-microbial agents at a final concentration of 0.04% (w/V), and stored at 4 ºC until use.
Avidin-Beads 2nd Ab-Biotin
3.1
Results and discussion Optimization of system parameters
The combination of antigen and antibodies depends on the complementarity between the two kinds of molecular structure and affinity in immunoreactions, and the required reaction time is influenced by a variety of factors and reaction conditions, meanwhile the efficiency of the immune response in microfluidic system is highly influenced by flow rate in microchannels and the surface tension on the chip. In the experiment, the immune reactions and rinsing processes were realized by the means of liquid flowing in microfluidic channels driven by syringe micropump. The f analysis conditions of injection rates and immune reaction times of reaction reagents, as well as rinsing conditions on the surface of the chip were optimized. Due to the volumes of the cartridge
Rinsing Buffer Sample
Waste Cartridge
AvidinBeads
BSA (negative control) Anti-AFP Anti-CEA Anti-CA19-9
2nd AbBiotin Rinsing Buffer
Chip
PDMS Layer Microchannel above the chip
PCB with MTJ chip
Sample Waste
Fig.3 Design sketch of MTJ Biosensor test card (A) and the fluidic channel (B)
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
chambers were fixed, the injection speed and rinsing conditions were determined by measuring the reagent injection times and rinsing buffer injection time. The experiments were designed respectively for three kinds of digestive system tumor markers, and only the optimization of the system parameters for CEA analysis was listed below. First, the concentrations of 2nd Ab-Biotin and Avidin-Beads were discussed when moderate microfluidic conditions and sufficient reaction time (15 min per step) were set, and the optimized concentrations of 2nd Ab-Biotin and Avidin-Beads were respectively 20 g mL–1 and 1 mg mL–1 (diluted by 10 times). After the volume of the rinsing buffer was set as 80 L, a series of injection times of rinsing buffer were employed to study the impact of the rinsing speed on specific reaction (with the concentrations of CEA of 1 ng mL–1) and nonspecific adsorption (without CEA) of antigen and antibody. As shown in Fig.4A, the number of nonspecific adsorbed magnetic particles decreased slightly with the shortening rinsing time, and the same trend was observed for the number of specific reacted magnetic particles. The optimal rinsing conditions for injection was to inject 80 L rinsing buffer in 60 s, by which a high signal to noise ratio was obtained. Immune response on the surface of the chip was shown in Fig.2. The interaction between antigens in the serum and antibodies on the chip was completed during the first incubation process in the cartridge, and the reaction efficiency of this step directly affected the formation of the final “Ab-Antigen-Ab” sandwich structure. Thus, the incubation time of the analyte with antigens had a direct and important influence on the immune analysis of tumor markers. Different static incubation times of analyte containing CEA were investigated under the concentration of 1 ng mL–1 CEA. The number of magnetic particles in the detected region increased
with the increase of incubation reaction time, and the increase in extent decreases after 10 min, roughly reaching a balance (Fig.4B). The static reaction times of 2nd Ab-Biotin and Avidin-Beads were also investigated. The reaction reached a general saturated status within 5 min. Therefore, the optimized reaction time for the three-step immune responses in the immune analysis of CEA was 10, 5 and 5 min, respectively. In microfluid bioprocessor, the system parameters were investigated for the analysis of AFP, CEA and CA19-9 respectively. The optimal reaction time of the three kinds of tumor markers were almost the same, while the optimal rinsing conditions were no difference (Table 1), so 60 s was identified as the optimal time in multi-target joint detection. Therefore, it could accomplish the detections of the above three tumor markers at the same time within 30 min by using the bioprocessor. 3.2
Joint detection of multiple digestive system tumor markers in magnetic bioprocessor
Based on the optimization of system parameters, different kinds of 1st Ab were coated on different regions of the chip, and the joint detections of three kinds of digestive system tumor markers AFP, CEA and CA19-9 were carried out. The standard working curves are shown in Fig.5. The Multi-target detection of a sample of three digestive system tumor markers could be finished within 30 min by the magnetic bioprocessor with the detection limits of 0.1 ng mL–1 for AFP, 0.1 ng mL–1 for CEA and 30 U mL–1 for CA19-9. The linear ranges of detection spanned four orders of magnitude, completely satisfying the requirements of clinical diagnostic. As shown in Fig.5, the standard working curves of three tumor markers were different in the low concentration
Fig.4 Optimization of rinsing time (A) and static reaction time (B) of CEA Table 1 Optimization of rinsing conditions in PDMS microchannels Reaction time of Antigen (min)
Optimized rinsing time of AFP (s)
Optimized rinsing time of CEA (s)
Optimized rinsing time of CA19-9 (s)
5
80
60
60
10
40
60
60
15
40
40
40
Optimized rinsing time of multi-targets (s) 60
LIAN Jie et al. / Chinese Journal of Analytical Chemistry, 2013, 41(9): 1302–1307
Fig.5
Standard curves for tumor markers by MTJ biosensor: detection of low-concentration (A. AFP, C.CEA, E. CA19-9) and high-concentration (B. AFP, D.CEA, F. CA19-9)
ranges and high concentration ranges, and the working curves all had good linearity (correlation coefficients close to 1.0). The deviations of analyzing samples of CEA are summarized in Table 2. The deviation was 11% at the concentration of 0.1 ng mL–1, 7.6% at the concentration of 1 ng mL–1, and as the concentration was above 1 ng mL–1, the deviation was less than 10%, meeting the clinical requirement for detection precision. Clinical serum samples were analyzed by the magnetic bioprocessor and the clinical used commercial ELISA kits. Correlation analysis was run between the detection results of two methods (Fig.6), and the correlation coefficients of two methods were 0.947 (CEA), 0.995 (AFP), 0.947 (CA19-9), respectively. The detection results of simultaneous analysis of three tumor markers by the magnetic bioprocessor agreed with ELISA results. By using the magnetic bioprocessor for the multi-biomarker test, the detection range and detection limit all reached the level of clinical use, with high sensitivity and rapid detection time of 30 min, which was much shorter than that of the conventional ELISA method (2–4 h per single target test). The optimal microfluidic reactions realized a series of biomarkers immune analysis; greatly reduced the time needed for the test process, and also realized the joint multi-target detection on the magnetic sensitive biosensors.
Fig.6 Correlation between three tumor markers in serum measured by MTJ biosensor and ELISA kit
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