Porous Hydrogel-Encapsulated Photonic Barcodes for Multiplex

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Porous hydrogel encapsulated photonic barcodes for multiplex detection of cardiovascular biomarkers Jingjing Ji, Wenbin Lu, Yi Zhu, Hong Jin, Yuyu Yao, Huidan Zhang, and Yuanjin Zhao ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00352 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Porous hydrogel encapsulated photonic barcodes for multiplex detection of cardiovascular biomarkers JingJing Jia,†, Wenbin Lua,†, Yi Zhua,†, Hong Jina, Yuyu Yaoa,*, Huidan Zhangc,*, Yuanjin Zhaoa,b,* aDepartment

of Cardiology, ZhongDa Hospital Affiliated with Southeast University, Nanjing, China

Email: [email protected] bState

Key Laboratory of Bioelectronics, school of Biological science and Medical Engineering.

Southeast University,Nanjing, China Email: [email protected] cSchool

of Engineering and Applied Sciences and Department of Physics, Harvard University,

Cambridge, Massachusetts 02138, United States Email: [email protected] †These authors contributed equally to this work

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Abstract: Early detection of cardiac troponin I (cTnI), B-type natriuretic peptide (BNP), and myoglobin (Myo) is essential for the diagnosis of acute myocardial infarction (AMI) and heart failure (HF). We designed a porous hydrogel-encapsulated photonic crystal (PhC) barcode-based suspension array for multiple cardiovascular marker detection. The hybrid hydrogel was composed of poly ethylene glycol diacrylate (PEGDA) and gelatin, resulting in a porous and hydrophilic scaffold which ensured stability of the PhC in aqueous solutions. The encapsulated PhC barcodes had stable diffraction peaks for the corresponding markers. Using a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of cardiovascular biomarkers in a single tube. The immunoassay results we tested on cTnI, BNP and Myo could be assayed in the ranges of 0.01 to 1000 ng/ml, 0.1 to 10000 pg/ml, and 1 to 10000 ng/ml with limits of detection of 0.009 ng/ml, 0.084 pg/ml, and 0.68 ng/ml at 3σ, respectively.

This method also showed acceptable accuracy and repeated detection, and

the results were consistent with the results of conventional clinical methods for detecting actual clinical samples. Therefore, suspension arrays based on hydrogel-encapsulated PhC barcodes is highly promising for AMI diagnosis. Keywords: AMI; Cardiovascular biomarkers; Multiplex assay; Hydrogel; Barcodes

Acute myocardial infarction (AMI) is one of the leading causes of deaths worldwide, in addition to being the most critical life-threatening condition [1, 2]. Early detection of cardiovascular markers like cardiac troponin I (cTnI), B-type natriuretic peptide (BNP) and myoglobin (Myo) can reduce morbidity and mortality among the patients. These markers can be easily detected in the patient sera using enzyme-linked immunosorbent assay (ELISA) [3, 4] and electro-chemiluminescence immunoassay (ECLIA). However, these conventional assays cannot simultaneously detect multiple markers, which lead to the wastage of precious biological materials, prolonged diagnosis and high costs. Furthermore, a single cardiac marker may not be effective as a diagnostic indicator of coronary heart disease (CAD) and heart failure (HF). Therefore, a composite high-throughput diagnostic platform is needed to detect multiple cardiovascular biomarkers simultaneously, and with high sensitivity. High-throughput assays are widely used for clinical diagnostics, gene expression studies, drug screening etc.[5-14], such as a new silver nanoshell silica photonic crystal bead (Ag-SPCB) SERS substrate can be prepared for the development of ultrasensitive multi-SERS bioassays and Raman signal amplification [15], which are broadly classified into planar and suspension arrays. The former include DNA/RNA and protein microarrays, which are limited by low-flexibility, slow reaction speed and low-reproducibility. Suspension arrays on the other hand consist of encoded microspheres that can detect specific targets; these “barcodes” can be chemical, physical, spectral, magnetic etc., and are decoded on the basis of the specific probes (e.g. fluorescent, luminescent etc.) immobilized on this substrate [16-18]. In recent years, barcodes have been developed using nanoparticles with unique optical/spectral characteristics, for e.g. photonic crystals (PhC) or dielectric nanostructures that limit


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propagation of light of a certain frequency range based on the band gap [19-25]. PhC-based barcodes embedded with fluorescent probes show remarkable stability, low signal background, and higher surface-to-volume ratio [26, 27], which allows for highly sensitive and accurate detection. In addition, combining spectrally distinct photonic barcodes can theoretically increase the target range and enable multiplex bioassays. However, these encoded-microspheres still have certain drawbacks, including slow diffusion of molecules to their binding sites and the inability to detect a high density of the target over a wide dynamic range. Therefore, it is essential to develop micro-carriers that can obviate the above limitations [28-31]. We designed PhC microcapsules consisting of a closely-packed opal PhC core encapsulated by inverse opal PhC-hydrogel shells with immobilized probes, as the respective encoding and sensing units. When used in a multiplex array, the PhC cores with stable diffraction peaks distinguish the probe-specific targets, and binding of the probes to the targets causes the shells to shrink, which is decoded as a corresponding blue shift in the Bragg diffraction peak position of PhC, and can be used for quantifying the amount of the target. The schematics are shown in Figure 1. In addition, the higher surface-to-volume ratios enable the loading of more fluorescent probes, which can further increase the sensitivity of the reaction. The sensitivity of our encapsulated PhC barcodes for multiple cardiovascular marker detection in complex media was in femtomolar scale. Taken together, we designed highly sensitive, flexible and high-throughput porous hydrogel encapsulated PhC barcodes for detecting and quantifying multiple cardiac biomarkers simultaneously from clinical samples, which is highly promising for AMI diagnosis. Materials and Methods Patients and samples A total of 20 AMI patients who were referred to the Chest Pain Center of the Zhongda Hospital affiliated to Southeast University of China from June 2018 to August 2018 were sequentially recruited, and serum samples were collected. The patients had been diagnosed on the basis of characteristic changes in their electrocardiogram (ECG), such as ST segment changes, new left bundle branch block, or Q waves changes in the motion of the heart wall, or demonstration of a thrombus in their angiograms, or by gross morphological changes seen at the autopsy. Patients with severe heart valve disease, coronary artery bypass grafting, cardiomyopathy, tumors or pregnancy was excluded. The samples were collected from consenting patients in accordance with the ethical and scientific guidelines of the Ethics Committee of Southeast University of China, who provided approval for the study (No: ZDKYSB194). Reagents Monodisperse silica nanoparticles of sizes 612, 525 and 475 nm were purchased from Nanjing Nanorian-bow Biotechnology Co. Ltd., and had been synthesized by the Stöber method. Mouse monoclonal antibodies against human cTnI (ab47003,1:1000), BNP (ab19645,1:1000) and Myo (ab77232,1:1000) were purchased from Abcam. Human cTnI (bs-10877P), BNP (bs-10879P), Myo (bs-10874P), and Cy3-conjugated goat anti-human cTnI antibody, anti-human BNP antibody and anti-human Myo antibody were obtained from Bioss. Bovine serum albumin (BSA) and phosphate buffer saline (PBS) were respectively purchased from Sigma-Aldrich (Shanghai, China) and Hyclone


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(China). The 2-Morpholinoethanesulfonic acid (MES) was purchased from Sigma Chemicals, and 1-ethyl-3-(3-dimethylaminepropyl) car-bodiimide (EDC) and N-hydroxysuccinimide (NHS) from Aladdin Reagent Co. Ltd. (Shanghai, China). Poly ethylene glycol diacrylate (PEGDA) of molecular weight 700 kDa and gelatin were purchased from Sigma-Aldrich (Shanghai, China). All other reagents were of analytical grade. Instruments The microfluidic device used for fabricating the silica colloidal crystal beads (SCCBs) was custom-made. All reactions were performed in flat tubes inside a constant temperature oscillator (Thermomixer comfort 5355, Eppendorf, Germany). The microstructures of SCCBs and the encapsulated PhC barcodes were observed by a scanning electron microscope (SEM, S-300N, Hitachi, Japan). The beads were imaged by a metalloscope (BX51, Olympus, Japan) equipped with a CCD camera (MP5.0, MediaCybernetics Evolution). The reflection spectra of the different particles were recorded by the same microscope equipped with a fiber optic spectrometer (HR2000, Ocean Optics, USA). The fluorescence intensities were detected by a fluorescence microscope (BX53,Olympus, Japan). Preparation of porous hydrogel-encapsulated PhC barcodes SCCBs of different sizes, corresponding to the reflection peaks of 612 nm, 525 nm and 475 nm, were prepared from silica nanoparticles by the droplet template method. Briefly, a 20% dispersion (w/v) of silica nanoparticles were broken into droplets containing independent particles by silicone oil flow in a microfluidic device[32]. The flow rates of the continuous phase and the dispersed phase were 10 and 0.5 ml/h respectively. The droplet suspension was incubated overnight at 75°C, and the silicone oil was washed with n-hexane. The beads were then calcined at 800°C for 3 hours to increase their mechanical strength. The hydrogel was prepared by mixing 20% PEG-DA with 13% gelatin in deionized water at room temperature, followed by the addition of 1% HMPP as the photo-initiator. The dried SCCBs were immersed in the pre-gel solution for 30 minutes to allow it to penetrate and fill the crystal lattices, followed by ultraviolet exposure for 15 seconds to polymerize the pre-gel. The hydrogel was washed with deionized water, and the surface particles were etched with 2% hydrofluoric acid (HF) to obtain microcapsules consisting of a PhC core enclosed by a porous hydrogel-PhC shell. Probe immobilization The PhC barcode shells were incubated with MES, EDC and NHS for 30 minutes with constant mixing, and washed with PBS to remove excess impurities. The different barcode preparations were incubated with the respective antibodies targeting cardiovascular markers at 25°C for 12 hours, and then with BSA for 2 hours. Detection of biomarkers For single-marker detection, each functional probe-immobilized PhC barcode (5 particles per tube) was sequentially incubated with 1µl of the corresponding marker and 5 µl fluorescent-labeled antibodies at 25°C for 30 minutes each, and observed under a fluorescence microscope. After every reaction step, the particles were washed with PBS to remove unbound material. Multiplex assays using all three barcode particles and their corresponding markers and fluorescent antibodies in combination were also


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conducted. Each experiment was repeated at least three times to ensure the accuracy and reliability of the test. Statistical analysis The ECLIA readings were automatically analyzed with the Modular analytics (Roche). The correlation between ECLIA and suspension array method was evaluated by Spearman regression analysis using SPSS 19.0 software. Results and discussion Preparation of the core-shell Phc particles Photonic barcodes were prep ared by replicating SCCBs, and immersed in a hydrogel solution, which can fill lattice spaces of the nanoparticles through capillary action [33]. After the pre-gel solution was UV-polymerized, the external particles were selectively etched, leaving an inverse opal hydrogel shell. Therefore, the PhC barcode core has the same structure as its hydrogel hybrid shell. The surface of the SCCBs and the PhC barcode microcapsules were observed by SEM. As shown in Figure 2, the hydrogel shell is a porous scaffold with a hexagonal symmetry. We used PEGDA and gelatin as the scaffold materials since the former is non-toxic and tough, thus ensuring the stability of the core-shell structure, whereas gelatin can easily bind to various biomolecules through its hydroxyl groups27 and increase the surface area for target binding. Optical characterization of the core PhC barcode particles Depending on the size and dielectric properties of the constituent silica nanoparticles, the different SCCB templates each have a unique photonic band gap (PBG), which inhibits the transmission of photons of a specific wavelength range that are then reflected, resulting in unique reflection spectra. The PhC core particles showed vivid colors and characteristic reflection peaks, even after hydrogel encapsulation (Figure 3). The main reflection peak of the core PhC particles can be estimated by Bragg’s equation: λ = 1.633 dnaverage, where d is the distance between the centers of two neighboring nanoparticles or inverse pores and naverage is the average refractive index of the particles. Therefore, combining nanoparticles of different sizes resulted in multiple encoding PhC particles with distinct diffraction peaks and spectra. Hydrogel encapsulation altered the spectral properties of the PhC particles; with longer etching time and increasing thickness (Figure S1a), two distinct reflection peaks appeared (Figure S1b), which resulted in a heterogeneous mix of encoding particles. Furthermore, although it is theoretically possible to increase the number of encoding PhC particles with different spectra, the peak from the hydrogel changes upon etching and could result in erroneous decoding. On the other hand, very thin shells decreased the structural stability of the microcapsules. The shells with optimal thickness were transparent with a stable reflection peak (Figure S2), and obtained after 20 min of etching (Figure S2b). Optimization of the PhC barcode shells and assay conditions The effect of the PhC microcapsule scaffold on barcode function was determined by analyzing the marker detection abilities of the 525 nm PhC-hydrogels with varying PEGDA or gelatin concentrations. The maximum fluorescence intensity was seen with 20% PEGDA (Figure 4a) and


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10-13% gelatin (Figure 4b), while keeping the concentration of the other polymer constant. However, 10% gelatin adhered weakly to the PhC nanoparticles, resulting in mechanically weak structures with insufficient optical emission. Therefore 20% PEGDA and 13% gelatin were used to synthesize the PhC barcode shells. We also observed a time-dependent increase in fluorescence intensity, peaking after 25 minutes of incubation (Figure 4c). The suspension array reaction was observed under a laser scanning confocal microscope, which showed a uniform and complete coating of the hydrogel shell with the markers (Figure 5). Since the immune reaction occurred within the porous hydrogel shells, it could not emit enhanced fluorescence due to the high optical transmission of the PhCs. This indicates that multiple barcodes can be used without any interference from the fluorescence signals, which is essential for multiplex detection. Detection of cardiovascular markers in serum samples After optimizing the assay conditions and PhC barcode shells, we tested the suspension array on 0.01 to 1000 ng/ml cTnI, 0.1 to 10000 pg/ml BNP, and 1 to 10000 ng/ml Myo. As shown in Figure 6, the fluorescence intensities increased gradually in a dose-dependent manner, although not linearly. The limits of detection for cTnI, BNP and Myo at 3σ using the PhC barcodes were 0.009 ng/ml, 0.084 pg/ml and 0.68 ng/ml respectively, whereas the clinical cut-off values of these markers are 0.01 ng/ml, 2 pg/ml and 23 ng/ml respectively. Therefore, our suspension array was more sensitive and had a wider detection range. Cross-reaction is a critical factor affecting the reliability of multiplex arrays. In this study ,three biomarkers, cTnI , BNP and Myo ,should be mixed and detected in one flat-bottom test tube,and the cross reactivity would influence the reliability of the multiplex immunoassay. To determine any cross-reactivity between the three cardiovascular markers, we evaluated the fluorescence intensities of the reactions with the concentration of one analyte constant (100 ng/ml) and varying that of the other two (100-500 ng/ml). As shown in Figure 6d, the uncertainty before and after the maximum change in fluorescence intensity was 5%, indicating negligible cross-reactivity. Clinical validation of the Phc barcodes To verify the analytical reliability and potential of the PhC barcode multiplex immunoassay, it was compared to standard ECLIA. We analyzed serum samples from 20 AMI patients at the Zhongda Hospital, and the results are shown in Figure 7. The linear regression equations between ECLIA (x-axis) and PhC barcodes (y-axis) are as follows: cTn1 – y = -0.74093+0.93922×(r2=0.95761), P < 0.001 BNP – y = 21.90927+0.85018 ×(r2=0.85018), P < 0.001 Myo – y = -24.496+1.0286×(r2=0.97629), P < 0.001 Despite lack of absolute correlation with ECLIA, PhC barcode multiplex array is suitable for clinical applications due to its high sensitivity, rapid detection, simple procedure, and requirement for low amounts of clinical material. However, the fabrication of encapsulated PhC barcodes is complex and time-consuming, and needs further development and automation. To summarize, we designed a novel PEGDA/gelatin hybrid hydrogel-encapsulated PhC barcode detection system. The hydrogel shell was porous and hydrophilic, which enabled molecular diffusion via interconnected nano-channels, stability in aqueous solutions, and stable emission peaks encoding


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different markers. It was able to detect minute levels of cTnI, BNP and Myo accurately from serum samples, and the results were consistent with that of conventional clinical tests. Therefore, PhC barcodes are a promising diagnostic tool for early detection of AMI patients. Associated content Supporting Information The Supporting Information is available free of charge. Optical characterization of the core-shell PhC particles; Optical images of the core−shell PhC particles with a transparent shell layer structure. Conflicts of interest The authors declare no conflicts of interest in this work. Acknowledgements This work was supported by the National Natural Science Foundation of China (No 81670326) and the Youth Medical Talents Project of Jiangsu Province (No QNRC2016814).

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Porous Hydrogel-Encapsulated Photonic Barcodes for Multiplex

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