- Email: [email protected]
Down’s syndrome screening with hydrogel photonic barcodes
Accepted Manuscript Title: Down’s syndrome screening with hydrogel photonic barcodes Authors: Hui Xu, Jingyin Zhang, Yueshuang Xu, Huan Wang, Fanfan Fu, Qionghua Xu, Yunlang Cai PII: DOI: Reference:
S0925-4005(17)31745-8 http://dx.doi.org/10.1016/j.snb.2017.09.079 SNB 23170
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
8-5-2017 30-8-2017 13-9-2017
Please cite this article as: Hui Xu, Jingyin Zhang, Yueshuang Xu, Huan Wang, Fanfan Fu, Qionghua Xu, Yunlang Cai, Down’s syndrome screening with hydrogel photonic barcodes, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.09.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Down’s syndrome screening with hydrogel photonic barcodes
Hui Xu1, Jingyin Zhang1, Yueshuang Xu2, Huan Wang3, Fanfan Fu3, Qionghua Xu3, Yunlang Cai1,*
1
Department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.
2
Department of Hematology and Oncology (Key Department of Jiangsu Medicine), Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.
3
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Email: [email protected]
Highlights
Hydrogel photonic barcodes with different reflection peak for multiplex detection. Concentration-optimized poly ethylene glycol for biomolecules diffusing and reaction. Highly sensitivity and stability were showed in detection of Down’s syndrome.
Abstract: For giving birth to a healthy baby without Down’s syndrome, screening the level of alpha fetoprotein (AFP) and free-beta human chorionic gonadotropin (β-hCG) in maternal serum has been a routine examination for all gravidas. However, many assays
only detect one target once time, which requires large samples and is low-throughput and costly. Herein, we describe a sensitive and inexpensive multiplex detection assay based on inverse opal structure hydrogel barcodes with poly(ethylene glycol) diacrylate (PEG-DA), poly(ethylene glycol) (PEG) and acrylic acid (AA) hybrid components. The polymerized PEG-DA hydrogel could guarantee the stabilities of the inverse opal structure, while the AA could offer active carboxyl groups for the probe immobilization. In addition, the interconnected pores of the inverse opal structure caused by PEG could also provide channels for biomolecules diffusing and reaction into the voids of the barcodes. The immunoassay results of our detection on AFP and β-hCG showed acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detection of practical clinical samples. Thus, the new platform is apparently suitable for multiplex detection of Down’s syndrome.
Keywords: Down’s syndrome; multiplex assay; hydrogel; photonic crystal; barcode
1. Introduction Down’s syndrome, also known as trisomy 21, associated with all or part of a third copy of chromosome 21 in fetus [1,2], generally causes physical growth delays, characteristic facial features, and mild to moderate intellectual disability [1,3]. Some of these syndromes may lead to considerable complications threatening individuals’ lives. Thus, screening maternal serum markers for Down’s syndrome during the prenatal period has become an established part of obstetric practice, which is significant for giving birth to a healthy child or termination of pregnancy [4]. One of these tests is
screening alpha fetoprotein (AFP) combining free Beta human chorionic gonadotropin (β-hCG) in maternal serum during the second trimester to estimate the risk of Down’s syndrome [5–9]. A wide variety of methods have been already applied for detecting maternal serum markers clinically, such as radioimmunoassay, enzyme linked immunosorbent assay, electrochemiluminescence immunoassay, etc [10–12]. However, such diagnostic technologies used today share a same drawback of only detecting one biochemical marker at one time, which would waste more material, reagents and time while detecting multiple markers. Various techniques have been invented for multiplex analysis, including of microarrays, microfluidic chips, barcodes, and so on [13–19]. For example, a successfully popular approach is planar array, in which the probe molecules are fixed on a flat substrate and encoded by their arranged location. Although capable of significant impact on analysis of biological target macromolecules, the planar array was restricted by some of its disadvantages like low-flexibility, slow reaction speed and nonrepeatability, etc. Then, among those technologies, barcodes based suspension array becomes an attractive preference for multiplex assay due to their higher flexibility and faster reaction kinetics in solution [13]. Although the fluorescent or quantum dots barcodes were well used in suspension array, they still have some disadvantages, such as photobleaching during storage and the potential interference of encoding fluorescence with analyte-detection fluorescence [20]. In this point of view, photonic crystal (PhC) barcodes, employing their encoded information in characteristic reflection peaks originated from their photonic band gap
(PBG), have been expressed for applying in suspension array as alternative microcarriers [21-25]. They were featured with distinct advantages such as remarkable code stability, low fluorescent background and much higher surface-to-volume ratio. Those strongpoints make the photonic suspension array proper for high sensitive detection. However, there remain certain drawbacks introduced by the presence of the solid surface of the PhC barcodes, which are with dull surface chemistry and can only provide limited surfaces for the probe immobilization and target detection, that’s why the hydrogel PhC barcodes were generated [26-30]. In addition, the potential value of the PhC barcodes in screening Down’s syndrome remains unexplored. In this study, we present a new type of hydrogel PhC barcodes for the multiplex immunoassay of Down’s syndrome (Figure 1). The hydrogel barcodes were employing the mixture of poly (ethylene glycol) diacrylate (PEG-DA), poly (ethylene glycol) (PEG) and acrylic acid (AA) to construct their inverse opal scaffold structures [31-33]. The inverse opal structure of hydrogel PhC barcodes and the porous structure generated by PEG could offer channels for macromolecules diffusing and reaction among the voids of barcodes, while the AA in the scaffolds could offer active chemical groups for probe immobilization and target detection. It was demonstrated that the hydrogel PhC barcodes were with high accuracy, detection reproducibility, and simple manipulation in multiplex immunoassay of biochemical marker AFP and β-hCG during the Down’s syndrome screening.
2. Experimental section 2.1. Materials
Three kinds of monodisperse silica nanoparticles with the size of 212, 256 and 297 nm were purchased from Nanjing Nanorianbow Biotechnology Co., Ltd. Silicon oil was purchased from Yunuo Chemicals Ltd., China. Bovine serum albumin (BSA) was purchased from Sigma Chemicals. Poly (ethylene glycol) diacrylate (PEG-DA) with molecular weights of 700, poly (ethylene glycol) (PEG) and 2-hydroxy-2methylpropiophenone (HMPP) photoinitiator were purchased from Sigma-Aldrich, Shanghai, China. Acrylic Acid (AA) was obtained from Alfa Aesar (China) Chemical Co., Ltd., Shanghai, China. 2-Morpholinoethanesulfonic Acid (MES) was purchased from Sigma Chemicals. 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd., Shanghai, China. Human AFP, β-hCG, mouse monoclonal anti-human AFP antibody, anti-human β-hCG antibody, FITC labeled rabbit polyclonal anti-human AFP antibody and anti-human β-hCG antibody were purchased from Shanghai Ruiqi biological technology CO., Ltd. All buffers were self-prepared using water purified in a Milli-Q system (Millipore, Bedford, USA). Deionized water was used for all experiments. Clinical serum samples were obtained from Zhongda Hospital of Southeast University, Nanjing, China. The collection and processing of clinical serum samples of human were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences. 2.2. Instruments The microfluidic device used for SCCBs-generation was home-made. Each step of reaction was conducted in a centrifuge tube or flat tube on a constant temperature
shaker (Thermomixer comfort 5355, Eppendorf, Germany). The microstructures of SCCBs and the hydrogel photonic crystal barcodes were characterized by a scanning electron microscope (SEM, S-300N, Hitachi, Japan). Photographs of the three kinds of beads were taken by metalloscope (BX51, Olympus, Japan) equipped with a CCD camera (MP5.0, Media Cybernetics Evolution). Reflection spectra of three beads were recorded by metalloscope equipped with a fiber optic spectrometer (HR2000, Ocean Optics, USA). Fluorescence intensity of the hydrogel photonic crystal barcodes was recorded by a fluorescence microscope (BX53, Olympus, Japan). 2.3. Preparation of the hydrogel photonic crystal (PhC) barcodes The silica colloidal crystal beads were assembled by silica nanoparticles through droplet template method. Firstly, the monodisperse silica nanoparticles with concentration of 20% (w/v) was broken into droplets by the oil flow in the microfluidic device. The flow rates of the continuous and dispersed phases were 5 and 0.5 mL/h, respectively. Then the droplets were collected by container filled with silicon oil, after being heated for one night, the silica colloidal crystal beads were washed and dried, then calcined at 800 ◦C for 3h to improve their mechanical strength. To meet the demand of multiplex immunoassay, three kinds of SCCBs with reflection peak position at 466, 563, 653 nm, respectively, were generated for the template of fabricating inverse opal hydrogel PhC barcodes. The hydrogel was synthesized by mixing 20% PEG-DA with 40% PEG, 10% AA and 1%HMPP-photoinitiator in deionize water at room temperature. To synthesize the hydrogel PhC barcode particles, the dried silica colloidal crystal beads with different
color were immersed in pre-gel solution prepared before for half an hour. The pre-gel solution could fill the gaps of silica colloidal crystal beads fully. Then SCCBs infiltrated with pre-gel solution was exposed to ultraviolet light for about 10s to polymerize the pre-gel. Finally, after removing the pre-gel on the surface of all the barcodes, the inverse opal hydrogel PhC barcodes would be obtained by corroding the hydrogel-encapsulated PhC barcode with hydrofluoric acid (HF 5 vol %) for about an hour to remove the silica nanoparticles. 2.4. Probe immobilization. The anti-human AFP antibody and anti-human β-hCG antibody probes were immobilized on hydrogel PhC barcodes by covalent bonding method. The carboxyl in AA was activated by EDC/NHS solution to react with amino. After washing the barcodes with PBS buffer, two kinds of the barcodes with different reflection peak position at 563 and 653nm, were reacted with anti-human AFP and anti-human β-hCG antibody probes in PBS buffer solution at 25℃ for 12h, respectively. At last, the two barcodes with their separate probes were well obtained after being washed with buffer solution. 2.5. Detection of maternal serum markers. Symbolling different serum markers with different hydrogel PhC barcodes equipped with specific spectrum, the concentration of each serum marker in maternal serum will be characterized by the fluorescence intensity of corresponding antibody under positive fluorescence microscopy. For single analysis, before incubating the functionalized hydrogel PhC barcodes
with targets (diluted in PBS) for 30min, the unreacted active chemical groups on PhC barcodes should be passivated with 1% BSA PBS for 2 h. Then, FITC labeled rabbit polyclonal anti-human AFP antibody, anti-human β-hCG antibody (0.1mg/mL) were added into the test tubes separately and incubated for 30min. During all the incubation process, the test tubes were shaken at 25 °C. After that, the unbound FITC labeled antibody was washed away by buffer solution. The number of replicates at any concentration was 5, 10 μl serum markers and anti-serum marker antibody was added into the tube per bead. For multiplexed analysis, two kinds of hydrogel PhC barcodes with the reflection peak position at 470 and 592nm, immobilized with mouse monoclonal anti-human AFP antibody and anti-human β-hCG antibody respectively, were put into one test tube for reaction. After passivating barcodes with 1%BSA, the maternal serum was added and shaken for 30 min. After the hydrogel PhC barcodes were washed with PBS buffer, two FITC labeled rabbit anti-serum marker antibodies were added into the mixed solution and incubated for 30min. At last, the hydrogel PhC barcodes were washed with PBS buffer again. All steps were processed away from light at 25°C.
3. Results and discussion 3.1 Fabrication of the hydrogel PhC barcodes. In a typical experiment, the inverse opal hydrogel barcodes were fabricated by replicating silica colloidal crystal beads (SCCBs). These SCCBs templates, prepared from the assembly of monodisperse silica colloidal nanoparticles in microfluidic device after dehydration and calcination, were added and immersed into the pre-gel solution.
After the pre-gel go through the voids between the nanoparticles and filled the voids fully of the templates by the capillary force, exposed it to ultraviolet light and the pregel in and out of the templates would be polymerized. Finally, the hydrogel PhC barcodes were obtained by removing the pre-gel on the surface of the templates and etching the nanoparticles in the barcodes with hydrofluoric acid. The surface imaging of the silica colloidal crystal bead templates and the hydrogel PhC barcodes were observed by scanning electron microscope (SEM) to confirm the microstructure of the fabricated barcodes (Figure 2). It can be seen that the nanoparticles on the surface of the silica colloidal crystal beads mainly formed a hexagonal alignment, and the hydrogel PhC barcodes replicated from the SCCBs had a similar highly ordered three dimensional (3D) inverse opal structure and hexagonal symmetrical porous surface. This structure would provide a nanopatterned platform with larger surface area for highly efficient entrapping biomolecules, since the diameter of biomolecules like antibody and antigen is about 12 -14nm, and the diameter of the interconnected pores in hydrogel PhC barcodes is more than 200nm, it is spaciously enough for serum molecules diffusing into this biochip. To complete the function of the photonic barcodes, we employed the mixture of poly (ethylene glycol) diacrylate (PEG-DA), poly (ethylene glycol) (PEG) and acrylic acid (AA) as the scaffold material for the hydrogel PhC barcodes. PEG-DA, as one type of hydrogel, equipped with some biological characteristics such as non-toxicity, high mechanical strength and low adhesion to protein, can guarantee the stability of the inverse opal structure and eliminate the nonspecific binding of proteins to meet the
demand of specific biomolecule screening. PEG, employed as the porogenic agent, capable with great biocompatibility and non-immunogenicity, can shape multiple nanopores among the hydrogel of PhC barcodes to provide more surface areas and channels for biomolecules diffusing and probes binding to carboxyl group provided by AA easily. The coded reflection peak of the PhC barcodes mostly depends on its structural periodicity. Because of its ordered nanostructure, the inverse opal hydrogel PhC barcodes is imparted with a photonic band gap (PBG) property and shows the corresponding structural color or characteristic reflection peak. Under normal incidence, the peak positions λ of the PhC barcodes can be estimated by Bragg’s equation: λ = 1.633dnaverage
(1)
Where d is the center-to-center distance between two neighboring nanopores, and naverage is the average refractive index of the PhC barcodes. As the naverage of our PhC barcodes is constant when the composed hydrogel was fixed, the peak value λ mainly depends on the diameter of the monodisperse silica colloidal nanoparticle. Therefore, a series of PhC barcodes with different reflection peak and colors could be obtained by changing the diameters of the silica nanoparticles and the derived macropores. Figure 3 showed the reflection images and spectra of these silica colloidal crystal beads with reflection peak positions at about 653nm, 563nm and 466nm and the corresponding hydrogel PhC barcodes, respectively. These PhC barcodes showed vivid structure colors of red, green and blue. It was found that after replicating these SCCBs with the hydrogel of PEG-DA and PEG, the reflection peak of the achieved hydrogel
PhC barcodes has changed to some extent. According to Bragg’s Law, the reflection peak was changed due to the change of naverage, because the average refractive index of the PhC barcodes has been changed along with the hydrogel taking the place of silica colloidal crystal beads. But the hydrogel PhC barcodes still possessed their distinctive reflection peak positions, and could be used as code elements for multiplex detection. Furthermore, the hydrogel PhC barcodes did not code with other agents, there is no need to worry about chemical instability and the fluorescence background. 3.2 Optimization of the hydrogel PhC barcodes. During the fabrication of hydrogel PhC barcodes, the different concentration of both PEG-DA and PEG would influence the final quality of the barcodes. Barcodes with lower concentration of PEG-DA showed poor mechanical properties, poor reflection peaks to code, and strong adhesion to each other, while higher concentration of PEG-DA shaped with nanopores too narrow for biomolecules diffusion. Therefore, five different concentration of PEG-DA were setting for synthesizing the hydrogel PhC barcodes based on the SCCBs with reflection peak position at 653nm to decide the most suitable one. By detecting one of the serum markers with barcodes composed of different concentration of the PEGDA, the hydrogel PhC barcodes composed of 10% PEG-DA exhibited strongest fluorescence intensity，and the 20% one in the next place (Figure 5a). In consideration of the mechanical properties, inferior adhesion and structural color of the barcodes，the 20% PEG-DA was finally chose to fabricate all the hydrogel PhC barcodes for the following experiment. The same was did to the PEG, while the
concentration of PEG-DA was remain unchanged, seven different concentration of PEG were setting for synthesizing the hydrogel PhC barcodes based on one type of the SCCBs with reflection peak at 653nm. Apparently, the reflection spectra were gradually changed along with the average refractive index of the hydrogel altering by different concentration of PEG (Figure 4). The higher concentration of PEG porogen would bring on more interconnected pores for biomolecules diffusing into the barcodes, but the mechanical strength of the hydrogel was decreased and became difficult to maintain, so the barcodes with 40% concentration of PEG did show the strongest fluorescence intensity (Figure5b). 3.3 Detecting serum markers. As the most common chromosomal disease, the natural morbidity of Down’s syndrome was detected with 1/1000-1/800, detection of maternal serum markers is very convenient and causes little trauma with taking little of blood from subjects. Also, comparing to other examinations, detecting serum markers is safer, cheaper and more accepted by people. AFP and β-hCG, the most two correlated serum markers with Down’s syndrome, were chosen to be screened in this study. Under optimal conditions, we detected routine samples of these two maternal serum markers with different concentrations ranging from 0.1 to 1000ng/ml. Figure 6 showed the dose-response curves for single detection of AFP and β-hCG. It was found that although the curves were not linear, the fluorescence intensity became higher as the concentration of analyte was increased from 1ng/ml to 1000ng/ml. The highest test values of AFP and β-hCG in clinical diagnosis are both under 200ng/ml, so the
detection ranges of our experiment were enough for practical application. Cross-reactivity is important for reliability of multiplex immunoassay. In this study, two maternal serum markers, AFP and β-hCG, should be mixed and detected in one flat-bottom test tube, because the cross reactivity would influence the reliability of the multiplex immunoassay. The cross reactivity in this research was evaluated with the fluorescence signals which were detected when the concentration of the specific analyte was constant while the concentration of another one was increased, respectively. Figure 7 showed the fluorescence intensities for specific serum markers at a concentration of 50ng/ml while the concentration of another increased to 200ng/ml. It could be seen that the maximum changes of the fluorescence intensities for each didn’t go beyond 5%, respectively. The results recommended that the cross-reactivity between AFP andβhCG could be neglected and we could realize the multiplex immunoassay. 3.4 Clinical detection of the hydrogel PhC barcodes. To check the application potential and the reliability of the hydrogel PhC barcodes for multiplex assay in clinic, we compared this method with the common method in clinic laboratory called electrochemiluminescence immunoassay (ECLIA). We examined almost 12 serum samples which were centrifuged from blood cells from 12 patients drawn by using the standard venipuncture technique in Zhongda Hospital, China. Figure 8 showed the results. The regression equations (linear) for these data were as followed (x axis, ECLIA; y axis, PhC barcodes): y =-3.84762+1.06339× (r2=0.98723) for AFP for Down’s syndrome. y =0.65616+0.96414× (r2=0.97761) for β-hCG for Down’s syndrome.
These data indicated that there was no significant difference for the detection results between the two methods, which meant our method had good reliability and application potential. In addition, this method could realize the multiplex immunoassay of the two serum markers in one test tube and needed fewer samples than ECLIA.
4. Conclusion. In conclusion，we have developed a novel hydrogel PhC barcode for multiplex detection of Down’s syndrome. The inverse opal hydrogel PhC barcodes replicated from SCCBs were constructed with the PEG-DA, PEG and AA. The polymerized PEDDA hydrogel guaranteed the stabilities of the inverse opal structure, and the interconnected pores of the inverse opal structure provided channels for biomolecules diffusing into the voids of whole barcodes. In addition, the PEG could shape porous structure to provide more channels for molecules diffusing and probes binding to carboxyl groups offered by AA. These advantages of the hydrogel PhC barcodes make them excellent suspension array. After optimizing the detection condition, the immunoassay results of our detection on AFP and β-hCG did show acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detections of practical clinical samples, thus, the hydrogel PhC barcodes is potential for multiplex detection of serum markers.
Acknowledgement This research was supported by Jiangsu Provincial Maternal and child health research project of Jiangsu Health Department (F201351).
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Hui Xu* received the Bachelor of Medicine degree from Yangzhou University in 2015. Now she is a graduate student at School of Medicine, Southeast University, Nanjing, China. Her current research is focused on biomarkers detection with biomaterials. Jingyin* Zhang is now both gynecologist and obstetrician in department of Obstetrics and Gynecology, Zhongda Hospital. She received her Bachelor of Medicine degree and Master of Medicine degree from Southeast University. Currently her research is focused on maternal and fetal medicine. Yueshuang Xu received Bachelor of Medicine degree (2013) from Southeast University, now she is pursuing the M.D. degree in Southeast University, Nanjing, China. Her research is focused on biomarkers detection with biomaterials. Huan Wang received the B.S. degree (2014) from Southeast University, Nanjing, China. Now he is pursuing the Ph.D. degree in the State Key Laboratory of Bioelectronics, Southeast University. His research is focused on protein-responsive photonic crystal. Fanfan Fu received the B.S. degree (2012) from School of Chemistry and Chemical Engineering, Shangrao Normal College. Now he is pursuing the Ph.D. degree at the State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China. His research is focused on photonic crystal biomaterials. Qionghua Xu received the B.S. degree from Xi’An Technology University in 2014, Xi’An, China. Now she is pursuing the Master of Science degree at the State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China. Her research is focused on microfluidic-based materials fabrication. Yunlang Cai is currently the chief physician and the director of department of Obstetrics and Gynecology, Zhongda Hospital. He received his B.S. degree from the Nanjing Railway Medical College (before) in 1987 and his M.D. degree in Medicine at the Southeast University in 2014. Dr. Cai worked in department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, since 1987. His current scientific interests are focused on diagnosis and treatment of gynecological and obstetric diseases. *
These authors contributed to the work equally and should be regarded as co-first
authors.
Figure 1. (a) Schematic of the procedure of single detection based on the hydrogel photonic crystal barcodes, carboxyl group from AA help antibodies linking to the hydrogel photonic crystal barcodes, which was constructed by PEG-DA. (b) Schematic of the barcode particles and the procedure of multiplex detection. Figure 2. (a) SEM image of the surface of silica colloidal crystals bead. (b, c) SEM images of the surface and inner 3D structure of inverse opal hydrogel photonic crystal barcodes with interconnected pores. Scale bars of the (a, b, c,) are 500 nm, 500nm and 1000nm separately. Figure 3. (a, d, g) Reflection images of three kinds of silica colloidal crystals beads with different structural colors; (b, e, h) Reflection images of the corresponding hydrogel photonic crystal barcodes; (c, f, i) Reflection spectra of three kinds of SCCBs and the corresponding hydrogel PhC barcodes with reflection at 653nm/588nm, 563nm/511nm and 466nm/446nm. Scale bars are 100 μm. Figure 4. Reflection spectra and reflection images change with different concentration of PEG in the hydrogel photonic crystal barcodes. Figure 5. (a) The relationship of fluorescence Intensity and the concentration of PEGDA; (b) The relationship of fluorescence Intensity and the concentration of PEG. 20% concentration of PEG-DA and 40% concentration of PEG was chose for constructing the barcodes. Figure 6. The fluorescence intensity increased with the exponentially upgrade concentration of AFP and β-hCG. Figure 7. This graph showing a cross-reactivity between AFP and β-hCG: One serum marker was detected at the concentration of 50ng/ml while the concentration of another
serum marker changing from 0ng/ml to 200ng/ml. Fluorescence intensity approximately remain unchanged. Standard deviations showing on the error bars. Figure 8. Comparing the detection by the hydrogel photonic barcodes and the standard ECLIA with 15 serum samples. The number of replicates for each clinical sample detection was five. Error bars represent standard deviations.
S0925-4005(17)31745-8 http://dx.doi.org/10.1016/j.snb.2017.09.079 SNB 23170
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
8-5-2017 30-8-2017 13-9-2017
Please cite this article as: Hui Xu, Jingyin Zhang, Yueshuang Xu, Huan Wang, Fanfan Fu, Qionghua Xu, Yunlang Cai, Down’s syndrome screening with hydrogel photonic barcodes, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.09.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Down’s syndrome screening with hydrogel photonic barcodes
Hui Xu1, Jingyin Zhang1, Yueshuang Xu2, Huan Wang3, Fanfan Fu3, Qionghua Xu3, Yunlang Cai1,*
1
Department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.
2
Department of Hematology and Oncology (Key Department of Jiangsu Medicine), Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.
3
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Email: [email protected]
Highlights
Hydrogel photonic barcodes with different reflection peak for multiplex detection. Concentration-optimized poly ethylene glycol for biomolecules diffusing and reaction. Highly sensitivity and stability were showed in detection of Down’s syndrome.
Abstract: For giving birth to a healthy baby without Down’s syndrome, screening the level of alpha fetoprotein (AFP) and free-beta human chorionic gonadotropin (β-hCG) in maternal serum has been a routine examination for all gravidas. However, many assays
only detect one target once time, which requires large samples and is low-throughput and costly. Herein, we describe a sensitive and inexpensive multiplex detection assay based on inverse opal structure hydrogel barcodes with poly(ethylene glycol) diacrylate (PEG-DA), poly(ethylene glycol) (PEG) and acrylic acid (AA) hybrid components. The polymerized PEG-DA hydrogel could guarantee the stabilities of the inverse opal structure, while the AA could offer active carboxyl groups for the probe immobilization. In addition, the interconnected pores of the inverse opal structure caused by PEG could also provide channels for biomolecules diffusing and reaction into the voids of the barcodes. The immunoassay results of our detection on AFP and β-hCG showed acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detection of practical clinical samples. Thus, the new platform is apparently suitable for multiplex detection of Down’s syndrome.
Keywords: Down’s syndrome; multiplex assay; hydrogel; photonic crystal; barcode
1. Introduction Down’s syndrome, also known as trisomy 21, associated with all or part of a third copy of chromosome 21 in fetus [1,2], generally causes physical growth delays, characteristic facial features, and mild to moderate intellectual disability [1,3]. Some of these syndromes may lead to considerable complications threatening individuals’ lives. Thus, screening maternal serum markers for Down’s syndrome during the prenatal period has become an established part of obstetric practice, which is significant for giving birth to a healthy child or termination of pregnancy [4]. One of these tests is
screening alpha fetoprotein (AFP) combining free Beta human chorionic gonadotropin (β-hCG) in maternal serum during the second trimester to estimate the risk of Down’s syndrome [5–9]. A wide variety of methods have been already applied for detecting maternal serum markers clinically, such as radioimmunoassay, enzyme linked immunosorbent assay, electrochemiluminescence immunoassay, etc [10–12]. However, such diagnostic technologies used today share a same drawback of only detecting one biochemical marker at one time, which would waste more material, reagents and time while detecting multiple markers. Various techniques have been invented for multiplex analysis, including of microarrays, microfluidic chips, barcodes, and so on [13–19]. For example, a successfully popular approach is planar array, in which the probe molecules are fixed on a flat substrate and encoded by their arranged location. Although capable of significant impact on analysis of biological target macromolecules, the planar array was restricted by some of its disadvantages like low-flexibility, slow reaction speed and nonrepeatability, etc. Then, among those technologies, barcodes based suspension array becomes an attractive preference for multiplex assay due to their higher flexibility and faster reaction kinetics in solution [13]. Although the fluorescent or quantum dots barcodes were well used in suspension array, they still have some disadvantages, such as photobleaching during storage and the potential interference of encoding fluorescence with analyte-detection fluorescence [20]. In this point of view, photonic crystal (PhC) barcodes, employing their encoded information in characteristic reflection peaks originated from their photonic band gap
(PBG), have been expressed for applying in suspension array as alternative microcarriers [21-25]. They were featured with distinct advantages such as remarkable code stability, low fluorescent background and much higher surface-to-volume ratio. Those strongpoints make the photonic suspension array proper for high sensitive detection. However, there remain certain drawbacks introduced by the presence of the solid surface of the PhC barcodes, which are with dull surface chemistry and can only provide limited surfaces for the probe immobilization and target detection, that’s why the hydrogel PhC barcodes were generated [26-30]. In addition, the potential value of the PhC barcodes in screening Down’s syndrome remains unexplored. In this study, we present a new type of hydrogel PhC barcodes for the multiplex immunoassay of Down’s syndrome (Figure 1). The hydrogel barcodes were employing the mixture of poly (ethylene glycol) diacrylate (PEG-DA), poly (ethylene glycol) (PEG) and acrylic acid (AA) to construct their inverse opal scaffold structures [31-33]. The inverse opal structure of hydrogel PhC barcodes and the porous structure generated by PEG could offer channels for macromolecules diffusing and reaction among the voids of barcodes, while the AA in the scaffolds could offer active chemical groups for probe immobilization and target detection. It was demonstrated that the hydrogel PhC barcodes were with high accuracy, detection reproducibility, and simple manipulation in multiplex immunoassay of biochemical marker AFP and β-hCG during the Down’s syndrome screening.
2. Experimental section 2.1. Materials
Three kinds of monodisperse silica nanoparticles with the size of 212, 256 and 297 nm were purchased from Nanjing Nanorianbow Biotechnology Co., Ltd. Silicon oil was purchased from Yunuo Chemicals Ltd., China. Bovine serum albumin (BSA) was purchased from Sigma Chemicals. Poly (ethylene glycol) diacrylate (PEG-DA) with molecular weights of 700, poly (ethylene glycol) (PEG) and 2-hydroxy-2methylpropiophenone (HMPP) photoinitiator were purchased from Sigma-Aldrich, Shanghai, China. Acrylic Acid (AA) was obtained from Alfa Aesar (China) Chemical Co., Ltd., Shanghai, China. 2-Morpholinoethanesulfonic Acid (MES) was purchased from Sigma Chemicals. 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd., Shanghai, China. Human AFP, β-hCG, mouse monoclonal anti-human AFP antibody, anti-human β-hCG antibody, FITC labeled rabbit polyclonal anti-human AFP antibody and anti-human β-hCG antibody were purchased from Shanghai Ruiqi biological technology CO., Ltd. All buffers were self-prepared using water purified in a Milli-Q system (Millipore, Bedford, USA). Deionized water was used for all experiments. Clinical serum samples were obtained from Zhongda Hospital of Southeast University, Nanjing, China. The collection and processing of clinical serum samples of human were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences. 2.2. Instruments The microfluidic device used for SCCBs-generation was home-made. Each step of reaction was conducted in a centrifuge tube or flat tube on a constant temperature
shaker (Thermomixer comfort 5355, Eppendorf, Germany). The microstructures of SCCBs and the hydrogel photonic crystal barcodes were characterized by a scanning electron microscope (SEM, S-300N, Hitachi, Japan). Photographs of the three kinds of beads were taken by metalloscope (BX51, Olympus, Japan) equipped with a CCD camera (MP5.0, Media Cybernetics Evolution). Reflection spectra of three beads were recorded by metalloscope equipped with a fiber optic spectrometer (HR2000, Ocean Optics, USA). Fluorescence intensity of the hydrogel photonic crystal barcodes was recorded by a fluorescence microscope (BX53, Olympus, Japan). 2.3. Preparation of the hydrogel photonic crystal (PhC) barcodes The silica colloidal crystal beads were assembled by silica nanoparticles through droplet template method. Firstly, the monodisperse silica nanoparticles with concentration of 20% (w/v) was broken into droplets by the oil flow in the microfluidic device. The flow rates of the continuous and dispersed phases were 5 and 0.5 mL/h, respectively. Then the droplets were collected by container filled with silicon oil, after being heated for one night, the silica colloidal crystal beads were washed and dried, then calcined at 800 ◦C for 3h to improve their mechanical strength. To meet the demand of multiplex immunoassay, three kinds of SCCBs with reflection peak position at 466, 563, 653 nm, respectively, were generated for the template of fabricating inverse opal hydrogel PhC barcodes. The hydrogel was synthesized by mixing 20% PEG-DA with 40% PEG, 10% AA and 1%HMPP-photoinitiator in deionize water at room temperature. To synthesize the hydrogel PhC barcode particles, the dried silica colloidal crystal beads with different
color were immersed in pre-gel solution prepared before for half an hour. The pre-gel solution could fill the gaps of silica colloidal crystal beads fully. Then SCCBs infiltrated with pre-gel solution was exposed to ultraviolet light for about 10s to polymerize the pre-gel. Finally, after removing the pre-gel on the surface of all the barcodes, the inverse opal hydrogel PhC barcodes would be obtained by corroding the hydrogel-encapsulated PhC barcode with hydrofluoric acid (HF 5 vol %) for about an hour to remove the silica nanoparticles. 2.4. Probe immobilization. The anti-human AFP antibody and anti-human β-hCG antibody probes were immobilized on hydrogel PhC barcodes by covalent bonding method. The carboxyl in AA was activated by EDC/NHS solution to react with amino. After washing the barcodes with PBS buffer, two kinds of the barcodes with different reflection peak position at 563 and 653nm, were reacted with anti-human AFP and anti-human β-hCG antibody probes in PBS buffer solution at 25℃ for 12h, respectively. At last, the two barcodes with their separate probes were well obtained after being washed with buffer solution. 2.5. Detection of maternal serum markers. Symbolling different serum markers with different hydrogel PhC barcodes equipped with specific spectrum, the concentration of each serum marker in maternal serum will be characterized by the fluorescence intensity of corresponding antibody under positive fluorescence microscopy. For single analysis, before incubating the functionalized hydrogel PhC barcodes
with targets (diluted in PBS) for 30min, the unreacted active chemical groups on PhC barcodes should be passivated with 1% BSA PBS for 2 h. Then, FITC labeled rabbit polyclonal anti-human AFP antibody, anti-human β-hCG antibody (0.1mg/mL) were added into the test tubes separately and incubated for 30min. During all the incubation process, the test tubes were shaken at 25 °C. After that, the unbound FITC labeled antibody was washed away by buffer solution. The number of replicates at any concentration was 5, 10 μl serum markers and anti-serum marker antibody was added into the tube per bead. For multiplexed analysis, two kinds of hydrogel PhC barcodes with the reflection peak position at 470 and 592nm, immobilized with mouse monoclonal anti-human AFP antibody and anti-human β-hCG antibody respectively, were put into one test tube for reaction. After passivating barcodes with 1%BSA, the maternal serum was added and shaken for 30 min. After the hydrogel PhC barcodes were washed with PBS buffer, two FITC labeled rabbit anti-serum marker antibodies were added into the mixed solution and incubated for 30min. At last, the hydrogel PhC barcodes were washed with PBS buffer again. All steps were processed away from light at 25°C.
3. Results and discussion 3.1 Fabrication of the hydrogel PhC barcodes. In a typical experiment, the inverse opal hydrogel barcodes were fabricated by replicating silica colloidal crystal beads (SCCBs). These SCCBs templates, prepared from the assembly of monodisperse silica colloidal nanoparticles in microfluidic device after dehydration and calcination, were added and immersed into the pre-gel solution.
After the pre-gel go through the voids between the nanoparticles and filled the voids fully of the templates by the capillary force, exposed it to ultraviolet light and the pregel in and out of the templates would be polymerized. Finally, the hydrogel PhC barcodes were obtained by removing the pre-gel on the surface of the templates and etching the nanoparticles in the barcodes with hydrofluoric acid. The surface imaging of the silica colloidal crystal bead templates and the hydrogel PhC barcodes were observed by scanning electron microscope (SEM) to confirm the microstructure of the fabricated barcodes (Figure 2). It can be seen that the nanoparticles on the surface of the silica colloidal crystal beads mainly formed a hexagonal alignment, and the hydrogel PhC barcodes replicated from the SCCBs had a similar highly ordered three dimensional (3D) inverse opal structure and hexagonal symmetrical porous surface. This structure would provide a nanopatterned platform with larger surface area for highly efficient entrapping biomolecules, since the diameter of biomolecules like antibody and antigen is about 12 -14nm, and the diameter of the interconnected pores in hydrogel PhC barcodes is more than 200nm, it is spaciously enough for serum molecules diffusing into this biochip. To complete the function of the photonic barcodes, we employed the mixture of poly (ethylene glycol) diacrylate (PEG-DA), poly (ethylene glycol) (PEG) and acrylic acid (AA) as the scaffold material for the hydrogel PhC barcodes. PEG-DA, as one type of hydrogel, equipped with some biological characteristics such as non-toxicity, high mechanical strength and low adhesion to protein, can guarantee the stability of the inverse opal structure and eliminate the nonspecific binding of proteins to meet the
demand of specific biomolecule screening. PEG, employed as the porogenic agent, capable with great biocompatibility and non-immunogenicity, can shape multiple nanopores among the hydrogel of PhC barcodes to provide more surface areas and channels for biomolecules diffusing and probes binding to carboxyl group provided by AA easily. The coded reflection peak of the PhC barcodes mostly depends on its structural periodicity. Because of its ordered nanostructure, the inverse opal hydrogel PhC barcodes is imparted with a photonic band gap (PBG) property and shows the corresponding structural color or characteristic reflection peak. Under normal incidence, the peak positions λ of the PhC barcodes can be estimated by Bragg’s equation: λ = 1.633dnaverage
(1)
Where d is the center-to-center distance between two neighboring nanopores, and naverage is the average refractive index of the PhC barcodes. As the naverage of our PhC barcodes is constant when the composed hydrogel was fixed, the peak value λ mainly depends on the diameter of the monodisperse silica colloidal nanoparticle. Therefore, a series of PhC barcodes with different reflection peak and colors could be obtained by changing the diameters of the silica nanoparticles and the derived macropores. Figure 3 showed the reflection images and spectra of these silica colloidal crystal beads with reflection peak positions at about 653nm, 563nm and 466nm and the corresponding hydrogel PhC barcodes, respectively. These PhC barcodes showed vivid structure colors of red, green and blue. It was found that after replicating these SCCBs with the hydrogel of PEG-DA and PEG, the reflection peak of the achieved hydrogel
PhC barcodes has changed to some extent. According to Bragg’s Law, the reflection peak was changed due to the change of naverage, because the average refractive index of the PhC barcodes has been changed along with the hydrogel taking the place of silica colloidal crystal beads. But the hydrogel PhC barcodes still possessed their distinctive reflection peak positions, and could be used as code elements for multiplex detection. Furthermore, the hydrogel PhC barcodes did not code with other agents, there is no need to worry about chemical instability and the fluorescence background. 3.2 Optimization of the hydrogel PhC barcodes. During the fabrication of hydrogel PhC barcodes, the different concentration of both PEG-DA and PEG would influence the final quality of the barcodes. Barcodes with lower concentration of PEG-DA showed poor mechanical properties, poor reflection peaks to code, and strong adhesion to each other, while higher concentration of PEG-DA shaped with nanopores too narrow for biomolecules diffusion. Therefore, five different concentration of PEG-DA were setting for synthesizing the hydrogel PhC barcodes based on the SCCBs with reflection peak position at 653nm to decide the most suitable one. By detecting one of the serum markers with barcodes composed of different concentration of the PEGDA, the hydrogel PhC barcodes composed of 10% PEG-DA exhibited strongest fluorescence intensity，and the 20% one in the next place (Figure 5a). In consideration of the mechanical properties, inferior adhesion and structural color of the barcodes，the 20% PEG-DA was finally chose to fabricate all the hydrogel PhC barcodes for the following experiment. The same was did to the PEG, while the
concentration of PEG-DA was remain unchanged, seven different concentration of PEG were setting for synthesizing the hydrogel PhC barcodes based on one type of the SCCBs with reflection peak at 653nm. Apparently, the reflection spectra were gradually changed along with the average refractive index of the hydrogel altering by different concentration of PEG (Figure 4). The higher concentration of PEG porogen would bring on more interconnected pores for biomolecules diffusing into the barcodes, but the mechanical strength of the hydrogel was decreased and became difficult to maintain, so the barcodes with 40% concentration of PEG did show the strongest fluorescence intensity (Figure5b). 3.3 Detecting serum markers. As the most common chromosomal disease, the natural morbidity of Down’s syndrome was detected with 1/1000-1/800, detection of maternal serum markers is very convenient and causes little trauma with taking little of blood from subjects. Also, comparing to other examinations, detecting serum markers is safer, cheaper and more accepted by people. AFP and β-hCG, the most two correlated serum markers with Down’s syndrome, were chosen to be screened in this study. Under optimal conditions, we detected routine samples of these two maternal serum markers with different concentrations ranging from 0.1 to 1000ng/ml. Figure 6 showed the dose-response curves for single detection of AFP and β-hCG. It was found that although the curves were not linear, the fluorescence intensity became higher as the concentration of analyte was increased from 1ng/ml to 1000ng/ml. The highest test values of AFP and β-hCG in clinical diagnosis are both under 200ng/ml, so the
detection ranges of our experiment were enough for practical application. Cross-reactivity is important for reliability of multiplex immunoassay. In this study, two maternal serum markers, AFP and β-hCG, should be mixed and detected in one flat-bottom test tube, because the cross reactivity would influence the reliability of the multiplex immunoassay. The cross reactivity in this research was evaluated with the fluorescence signals which were detected when the concentration of the specific analyte was constant while the concentration of another one was increased, respectively. Figure 7 showed the fluorescence intensities for specific serum markers at a concentration of 50ng/ml while the concentration of another increased to 200ng/ml. It could be seen that the maximum changes of the fluorescence intensities for each didn’t go beyond 5%, respectively. The results recommended that the cross-reactivity between AFP andβhCG could be neglected and we could realize the multiplex immunoassay. 3.4 Clinical detection of the hydrogel PhC barcodes. To check the application potential and the reliability of the hydrogel PhC barcodes for multiplex assay in clinic, we compared this method with the common method in clinic laboratory called electrochemiluminescence immunoassay (ECLIA). We examined almost 12 serum samples which were centrifuged from blood cells from 12 patients drawn by using the standard venipuncture technique in Zhongda Hospital, China. Figure 8 showed the results. The regression equations (linear) for these data were as followed (x axis, ECLIA; y axis, PhC barcodes): y =-3.84762+1.06339× (r2=0.98723) for AFP for Down’s syndrome. y =0.65616+0.96414× (r2=0.97761) for β-hCG for Down’s syndrome.
These data indicated that there was no significant difference for the detection results between the two methods, which meant our method had good reliability and application potential. In addition, this method could realize the multiplex immunoassay of the two serum markers in one test tube and needed fewer samples than ECLIA.
4. Conclusion. In conclusion，we have developed a novel hydrogel PhC barcode for multiplex detection of Down’s syndrome. The inverse opal hydrogel PhC barcodes replicated from SCCBs were constructed with the PEG-DA, PEG and AA. The polymerized PEDDA hydrogel guaranteed the stabilities of the inverse opal structure, and the interconnected pores of the inverse opal structure provided channels for biomolecules diffusing into the voids of whole barcodes. In addition, the PEG could shape porous structure to provide more channels for molecules diffusing and probes binding to carboxyl groups offered by AA. These advantages of the hydrogel PhC barcodes make them excellent suspension array. After optimizing the detection condition, the immunoassay results of our detection on AFP and β-hCG did show acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detections of practical clinical samples, thus, the hydrogel PhC barcodes is potential for multiplex detection of serum markers.
Acknowledgement This research was supported by Jiangsu Provincial Maternal and child health research project of Jiangsu Health Department (F201351).
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Hui Xu* received the Bachelor of Medicine degree from Yangzhou University in 2015. Now she is a graduate student at School of Medicine, Southeast University, Nanjing, China. Her current research is focused on biomarkers detection with biomaterials. Jingyin* Zhang is now both gynecologist and obstetrician in department of Obstetrics and Gynecology, Zhongda Hospital. She received her Bachelor of Medicine degree and Master of Medicine degree from Southeast University. Currently her research is focused on maternal and fetal medicine. Yueshuang Xu received Bachelor of Medicine degree (2013) from Southeast University, now she is pursuing the M.D. degree in Southeast University, Nanjing, China. Her research is focused on biomarkers detection with biomaterials. Huan Wang received the B.S. degree (2014) from Southeast University, Nanjing, China. Now he is pursuing the Ph.D. degree in the State Key Laboratory of Bioelectronics, Southeast University. His research is focused on protein-responsive photonic crystal. Fanfan Fu received the B.S. degree (2012) from School of Chemistry and Chemical Engineering, Shangrao Normal College. Now he is pursuing the Ph.D. degree at the State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China. His research is focused on photonic crystal biomaterials. Qionghua Xu received the B.S. degree from Xi’An Technology University in 2014, Xi’An, China. Now she is pursuing the Master of Science degree at the State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China. Her research is focused on microfluidic-based materials fabrication. Yunlang Cai is currently the chief physician and the director of department of Obstetrics and Gynecology, Zhongda Hospital. He received his B.S. degree from the Nanjing Railway Medical College (before) in 1987 and his M.D. degree in Medicine at the Southeast University in 2014. Dr. Cai worked in department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, since 1987. His current scientific interests are focused on diagnosis and treatment of gynecological and obstetric diseases. *
These authors contributed to the work equally and should be regarded as co-first
authors.
Figure 1. (a) Schematic of the procedure of single detection based on the hydrogel photonic crystal barcodes, carboxyl group from AA help antibodies linking to the hydrogel photonic crystal barcodes, which was constructed by PEG-DA. (b) Schematic of the barcode particles and the procedure of multiplex detection. Figure 2. (a) SEM image of the surface of silica colloidal crystals bead. (b, c) SEM images of the surface and inner 3D structure of inverse opal hydrogel photonic crystal barcodes with interconnected pores. Scale bars of the (a, b, c,) are 500 nm, 500nm and 1000nm separately. Figure 3. (a, d, g) Reflection images of three kinds of silica colloidal crystals beads with different structural colors; (b, e, h) Reflection images of the corresponding hydrogel photonic crystal barcodes; (c, f, i) Reflection spectra of three kinds of SCCBs and the corresponding hydrogel PhC barcodes with reflection at 653nm/588nm, 563nm/511nm and 466nm/446nm. Scale bars are 100 μm. Figure 4. Reflection spectra and reflection images change with different concentration of PEG in the hydrogel photonic crystal barcodes. Figure 5. (a) The relationship of fluorescence Intensity and the concentration of PEGDA; (b) The relationship of fluorescence Intensity and the concentration of PEG. 20% concentration of PEG-DA and 40% concentration of PEG was chose for constructing the barcodes. Figure 6. The fluorescence intensity increased with the exponentially upgrade concentration of AFP and β-hCG. Figure 7. This graph showing a cross-reactivity between AFP and β-hCG: One serum marker was detected at the concentration of 50ng/ml while the concentration of another
serum marker changing from 0ng/ml to 200ng/ml. Fluorescence intensity approximately remain unchanged. Standard deviations showing on the error bars. Figure 8. Comparing the detection by the hydrogel photonic barcodes and the standard ECLIA with 15 serum samples. The number of replicates for each clinical sample detection was five. Error bars represent standard deviations.