Three-Dimensional Immunosensing Platform Based on a Hybrid

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Three-dimensional Immunosensing Platform Based on Hybrid Nanoflower for Sensitive Detection of Alpha-fetoprotein and Enterovirus 71 Yucheng Liu, Mingyuan Du, Jie Zhu, Xingwen Hu, Xinghu Ji, and Zhike He ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01109 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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ACS Applied Nano Materials

Three-dimensional Immunosensing Platform Based on Hybrid Nanoflower for Sensitive Detection of Alpha-fetoprotein and Enterovirus 71 Yucheng Liu†, Mingyuan Du†, Jie Zhu‡, Xingwen Hu‡, Xinghu Ji†, Zhike He*†

†

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡

Department of Pediatric Endocrine and Genetic Metabolic Disease, Maternal and

Children’s Hospital of Hubei Province, Wuhan 430072, China;

*Corresponding author. Tel: +86 27-68756557; E-mail: [email protected] (Z. He)

1

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ABSTRACT In this work, we constructed the nanoflower (NF) based three-dimensional (3D) immunosensing platform to improve the performance of conventional colorimetric immunoassay. To be specific, the biotinylated antibody-inorganic hybrid nanoflowers were prepared by encapsulating biotinylated antibody in inorganic nanocrystal composites. Then taking the advantage of the large amount of loading antibodies as well as the unique 3D structure of the hybrid nanoflower, we proposed the concept of “nanoflower based 3D immunosensing platform”. The nanoflower based 3D immunosensing platform exhibited high capture efficiency by providing the target antigens with significantly enhanced accessibility to capture antibodies, which provide us a new way to improve the sensitivity of immunosensor. In the determination of both protein biomarkers alpha-fetoprotein (AFP) and Human Enterovirus 71, the proposed 3D immunosensor exhibited outstanding performance, including longer-term stability, wider linear range, and enhanced sensitivity. Validated with clinical samples, the 3D immunosensor exhibits a great consistency with outcomes of current clinical polymerase chain reaction (PCR). Therefore, nanoflower based 3D immunosensing platform allows specific and versatile detection of diverse targets, and hence has a great potential in sensitive and early detection of various diseases.

KEYWORDS: hierarchical porous structures; immunosensor; colorimetric; protein biomarker; Enterovirus 71

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INTRODUCTION In modern medicine, early diagnosis and treatment of disease that allow dentification of diseases at an early stage and improve treatment efficiency, rely largely on development of modern analytical methods and techniques.1-3 Large number of approaches were developed for rapid and sensitive detection according to different kinds of analytes, including polymerase chain reaction (PCR),4 DNA sensor,5, electrochemical assay,7,

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colorimetric assay,9,

10

6

and surface plasmon-enhanced

spectroscopy.11 Among them, visual detection has attracted increasing attention for its allowing naked-eye readout assay of targets, especially in rural area with resource-constrained settings.5,

12

Being highly specific techniques, colorimetric

immunoassays are the most widely used detection approaches of clinically relevant biomarkers.12 However, conventional colorimetric immunoassay has many drawbacks. For example, antibodies immobilized on the surface of the plate by physical adsorption tend to be unstable because of antibodies’ desorption caused by the washing process and lose their activity.13 Apart from these, the most common problem that researchers concerned is the narrow linear range and poor sensitivity of the colorimetric immunoassay. Generally, the limit of conventional detection of ELISA are confined in nanomolar or picomolar range for two main reasons:

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(Ⅰ) lack of

powerful signal output and signal amplification strategy; (Ⅰ) poor capture ability for antigens resulted from a low ratio of capture antibodies, and inefficient accessibility of capture antibodies to antigens due to the two-dimensional surface of plate. Therefore, the sensitivity of the immunoassay can be improved from the two aspects.

On the one hand, a huge number of signal reporters and amplification strategies have been developed to boost the sensitivity of immunosensor, including gold nanoparticles as signal reporter,15 silver nanoparticles and MnO2 based signal tags,16 enzyme-based signal amplification,17 and assembly of nucleic acids and proteins.18 On the other hand, thanks to the advancement of nanotechnology,19-21 the unique structure of 3



nanomaterials provides new opportunity to further improve the sensitivity.

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Up to

date, various substrates have been specifically designed to improve the performance of immunoassays.22-24 Many materials, such as carbon nanotubes,7 zinc oxide nanorods,25 and even virus nanoparticles,1 have been employed to construct three-dimensional nanostructured substrates for their huge surface area and high loading capacity. However, complicated synthetic procedure and requirement of expensive instrument and time-consuming labors are usually required in these methods, hindering the extensive usage of this nanomaterials. Recently, a new type of hierarchical three dimensional nanostructure, organic-inorganic hybrid nanoflower made of protein and Cu3(PO4)2, has received significant investigative interests due to its facile and environmentally-friendly preparation (one-step coprecipitation method) and its artistic morphology with large surface-to-volume ratio.26, 27 Here, inspired by the three dimensional hierarchically structure of protein-inorganic hybrid nanoflower, we propose the concept of “NF based 3D ELISA”, and comprehensively demonstrate the great performance of the three-dimensional substrate formed by nanoflower. To the best of our knowledge, no related work has been reported about employing the hierarchical structure of hybrid nanoflower as 3D platform with high-efficiency capture ability in ELISA.

In this work, the hierarchical three-dimensional structure of organic-inorganic hybrid nanoflower was used to construct a 3D immunosensing platform. As illustrate in Scheme 1a, in the first step, biotinylated antibody was immobilized on the hybrid nanoflower through one-step mild preparing process without damaging the activity of antibody. This synthetic approach does not involve any toxic elements, extreme harsh conditions and complex synthesis procedure, and antibodies are immobilized on inorganic component through this facile bioinspired strategy without activity loss. Then through the biotin-streptavidin reaction, the biotinylated antibody-Cu3(PO4)2 hybrid nanoflower was conjugated to the immobilizer streptavidin to form the 3D immunosensing platform. As illustrated in Scheme 1b, in the presence of targets, the 4


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antibody nanoflower based 3D platform can recognize and capture the targets through the specific antigen-antibody reaction. At last, the captured targets can bind to the signal antibodies labelled by horseradish peroxidase (HRP), resulting in obvious color changes in the presence of tetramethylbenzidine (TMB) and H2O2. The proposed robust and versatile 3D immunosensing platform possesses the advantages of stability, rapidness, and enhanced sensitivity. More importantly, the hierarchical 3D structure of antibody- Cu3(PO4)2 hybrid nanoflower with high loading capacity of capture antibody provides more frequent antibody-marker binding sites to realize a high-efficiency capture of bioanalytes further boosting the sensitivity. The advantages above bring the proposed 3D immunosensing platform great potential for ultrasensitive and versatile detection of both AFP and EV71 in real clinical sample.

EXPERIMENTAL SECTION Reagents and materials All reagents and apparatus are provided in the Supporting Information

Synthesis and Characterization of antibody-Cu3(PO4)2 hybrid nanoflowers The antibody hybrid nanoflowers were prepared according to the literature with some modification.26 Typically, 4 µL of aqueous CuSO4 solution (120 mM) was added to 600 mL of PBS (0.1 mM, pH 7.4) containing a series of concentrations of biotinylated antibody (0.005 mg/mL to 0.5 mg/mL), respectively, followed by incubation at 25Ⅰ for 3 days at mild condition without stirring. The prepared nanoflower precipitate was collected through centrifugation (10000 rpm for 10 min) and washed with deionized water three times. And the characterization of antibody-Cu3(PO4)2 hybrid nanoflowers was provided in the supporting information.

Nanoflower based 3D immunosensing platform for AFP determination in standard buffer Briefly, AFP was prepared at different concentrations in the standard buffer (0.1-100 5



ng/mL). First, 50 µL of the as-prepared antibody (biotin)-Cu3(PO4)2 hybrid nanoflower was first attached to the bottom of Thermo Scientific Nunc Immobilizer Streptavidin at 37Ⅰ for 60 min. After washing with PBS-Tween buffer (all the binding steps described below were followed by a rinsing step), 2% BSA was used as blocking solution for 1h at 37 °C to prevent nonspecific adsorption. 50 µL of each dilution of the previously prepared solution of AFP in standard buffer was piped into each well of plates and incubated for 1 hour at 37Ⅰ. Then, the Ab-HRP (50µL, 0.02 mg/mL) was bound to AFP through an immunoreaction for 1 hour at 37Ⅰ. Finally, the ready-to-use TMB (50 µL) was added and oxidized by H2O2 for 5 min at 37Ⅰ, and 2M H2SO4 was added to end the change of color in the microplate. For the blue color of the hybrid nanoflower would affect the results of the assays, the 96-well plate was not placed into the microplate reader to get the results directly. Instead, to avoid the interference of the absorbance of hybrid nanoflower, the hybrid nanoflowers were remain in the bottle of the 96-well plate after finishing the catalytic reaction, and the supernatant is taken out for further UV absorption spectrometry. The supernatant was collected and diluted to 500 µL, and then the absorbance of the diluted supernatant at 450 nm was measured by UV-2250 spectrophotometer (Shimadzu, Japan).

Nanoflower based 3D immunosensing platform for protein biomarker AFP analysis in human serum The performance of the proposed immunosensing platform for protein biomarker determination was evaluated in real human serum sample. The same procedure in standard buffer was conducted for various concentration of AFP in 10-fold diluted human serum samples. 0.5, 1.0, 5.0, and 10.0 ng/mL of AFP in 10-fold diluted human serum sample (at least three replicates for each portion biomarker concentration) and the recovery percentage of protein biomarker AFP from human serum were calculated as compared with standard buffer.

Nanoflower based 3D immunosensing platform for EV71 detection in standard 6


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buffer Briefly, EV71 was prepared at different concentrations in the standard buffer (7.396*103 – 7.396*109 copies/mL). First, 50 µL of the as-prepared antibody (biotin)-Cu3(PO4)2 hybrid nanoflower was first attached to the bottom of Thermo Scientific Nunc Immobilizer Streptavidin at 37Ⅰ for 60 min. After washing with PBS-Tween buffer (all the binding steps described below were followed by a rinsing step), 2% BSA was used as blocking solution for 1h at 37 °C to prevent nonspecific adsorption. 50 µL of each dilution of the previously prepared solution of EV71 in standard buffer was piped into each well of plates and incubated for 1 hour at 37Ⅰ. Then, the Ab-HRP (50µL, 0.02 mg/mL) was bound to AFP through an immunoreaction for 1 hour at 37Ⅰ. Finally, the ready-to-use TMB (50 µL) was added and oxidized by H2O2 for 5 min at 37Ⅰ. After adding 2M H2SO4 to end the change of color in the microplate, the supernatant was collected and diluted to 500 µL. And then the absorbance of the diluted supernatant at 450 nm was measured by UV-2250 spectrophotometer (Shimadzu, Japan).

Nanoflower based 3D immunosensing platform for EV71 clinical sample analysis 10 clinical positive samples of hand, foot and mouth disease (HFMD) patients that suffered from EV71 infection was provided by Maternal and Children’s Hospital of Hubei Province. And the RT-PCR results of the clinical samples were also provided by Maternal and Children’s Hospital of Hubei Province. The same procedure in standard buffer was conducted for 5-fold dilution of the clinical samples.

RESULTS AND DISCUSSION Characterization of the antibody-Cu3(PO4)2 hybrid nanoflowers Biotinylated antibody-Cu3(PO4)2 hybrid nanoflower was prepared by adding CuSO4 to phosphate buffered saline (PBS) containing capture antibody at pH 7.4 and room temperature for three days. Scanning electron microscopy (SEM) and transmission electron microscope (TEM) images were collected to demonstrate the morphology of 7



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the antibody-Cu3(PO4)2 hybrid nanoflowers. As presented in Figure 1a and b, the antibody-Cu3(PO4)2

hybrid

nanoflowers

are

uniform

with

hierarchically

three-dimensional flower-like nanostructure. A high-resolution TEM image of single nanopetal is shown in Figure 1c，and the small spot in the nanopetal was the crystal lattice of copper phosphate, revealing the formation of Cu3(PO4)2. The diffraction peaks in X-ray diffraction (XRD) analysis agreed well with the feature peak of Cu3(PO4)2·3H2O according to the JCPDS card (PDF#022-0548), confirming the inorganic component in the hybrid nanoflowers (Figure 1d). The energy dispersive X-ray spectroscopy (EDS) experiments were conducted. EDS mapping (Figure 1e) revealed that five typical elements including O, C, N, Cu and P were observed in the nanoflower and attributed to Cu3(PO4)2/antibody nanocomposites. EDS spectrum (Figure 1f) revealed complete element distribution in the nanoflower. The encapsulation efficiency which can be defined as the ratio of the amount of immobilized antibody to the total amount of antibody was calculated to be 37.4% by using the Branford assay (see supporting information).

Stability test of the antibody-Cu3(PO4)2 hybrid nanoflowers The stability of antibody (biotin)-Cu3(PO4)2 hybrid nanoflower was further compared with that of free antibody (biotin). After 7 days of storage at 37Ⅰ in PBS, the hybrid nanoflower maintained approximately 95% of their capture ability, while the free antibody (biotin) only retained 63% of its initial activity (Figure 2a). Besides, the hierarchical three-dimensional structure of hybrid nanoflower had little change even for months of storage in PBS (Figure 2b). Therefore, the antibody (biotin)-Cu3(PO4)2 hybrid nanoflower exhibited outstanding advantage in the stability.

Advanced capture capacity of 3D nanoflower based immunosensing platform To investigate the advanced capture ability of the hierarchical 3D nanoflower based immunosensing platform, the performance tests were conducted to compare the capture ability of conventional 96-well flat plate with that of 3D nanoflower based 8


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platform. For the traditional ELISA usually involved tedious steps which may affect the accuracy of the investigation, the steps of ELISA were simplified to validate the impact of the substrate structure. Here, goat anti-mouse lgG (biotin) was employed as the capture antibody and HRP labelled mouse lgG was used as a typical antibody to evaluate the amount of antibody immobilized on the surface. For fair comparison between capture ability of conventional 96-well flat plate and that of 3D nanoflower based platform, the following factors were optimized: the concentration of free antibody in conventional 96-well flat plate, the concentration of antibody hybrid nanoflower in 3D nanoflower based platform, and the incubation time of the antibody hybrid nanoflower. As shown in Figure S1a, with the increase of the concentration of free antibody from 1.0 to 50.0 µg/mL, the absorbance kept increasing and reached the platform, and the 20 µg/mL of antibody is sufficient and saturated for further step. The different kinds of hybrid nanoflower were synthesized by introducing various concentrations of antibody. The absorbance increased when the concentration of antibody introduced into hybrid nanoflower increased from 5 to 50 µg/mL, which indicated that the amount of antibody immobilized in the hybrid nanoflower would impact the capture ability of 3D substrate (Figure S1b). To investigate the relationship between the structure of substrate and the capture ability, the number of antibodies in conventional 96-well flat plate and in nanoflower based platform were controlled at the same concentration in initial step. As depicted in Figure S1c, there was a detectable signal after 5 min, and the absorbance increased with the reaction time and then remain the same over 60 min. To ensure a rapid and stable detection, 60 min was as the optimized incubation time. Figure S1d exhibited capture ability of conventional 96-well flat plate and 3D nanoflower based platform at different reaction time with antibodies labelled HRP respectively, which indicated that the 3D hybrid nanoflower in 3D immunosensing platform has advantage over the conventional 96-well flat plate.

Nanoflower based 3D immunosensing platform for AFP determination 9



Possessing advantage of the rapid coating procedure and the unique hierarchically three-dimensional structure, the hybrid nanoflower based 3D substrate was used for rapid and high-efficiency immunoassay of disease-related biomarker, and AFP was used as the model protein in the work. The nanoflower based 3D ELISA were compared fairly with the conventional ELISA that usually performed in 2D scale. In nanoflower based immunosensing platform, the 96-well microplate modified with SA was coupled with the biotinylated antibody nanoflower rapidly through the SA-biotin reaction, while in conventional colorimetric immunoassay, the free AFP capture antibody was attached in the 96-well microplate through overnight physical absorption. Then the coated plates were blocked with BSA for 1 h at 37 Ⅰ. In the presence of the target protein, the captured protein was recognized by the Ab-HRP, which catalyzed the TMB into the oxidation product of TMB in the presence of H2O2, thus the color of the solution changing from clear to yellow after terminating with 2 M H2SO4. The changed color was readily measured by a UV-vis spectrophotometer or even can be observed with naked eye. For nanoflower based immunoassay, the whole process can be done within several hours, while the conventional colorimetric immunosensor takes a day and night. Ionic strength and pH value were first investigated. A series of pH ranging from 6.8 to 9.0 and different concentrations of NaCl were optimized, as shown in Figure S2, 0.01 M PBS with 150 mM NaCl (pH 7.4) was used for further detection of AFP. The detection range of the 3D ELISA was compared with that of the conventional ELISA. As shown in the Figure 3, the 3D ELISA can detect AFP as low as 0.1 ng/mL (Figure 3b), while the conventional colorimetric immunosensor was not able to detect the AFP when its concentration is lower than 5 ng/mL (Figure 3a). In the meantime, the result of 2D ELISA see no difference when the concentration is higher than 50 ng/mL, but the 3D ELISA can detect the high concentration of AFP from 10 ng/mL to 100 ng/mL as well. Therefore, nanoflower based 3D immunosensing platform has wider detection range than conventional colorimetric immunosensor.

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Sensitivity also is an important criterion to assess ELISA detection method. The sensitivity of the Sandwich ELISA is dependent on four factors: the number of molecules of the first antibody that are bound to the solid phase, the avidity of the first antibody for the antigen, the avidity of the enzyme labelled antibody for the antigen and the specific activity of the enzyme labelled antibody. As depicted in Figure S3, nanoflower based 3D immunosensor with large surface-to-volume ratio allows the immobilization of a greater amount of capture antibodies. Also, the 3D structure of hybrid nanoflower provides the immobilized capture antibodies with significantly high accessibility to analyte antigens from all sides. Here quantitative analysis was conducted by comparing the absorbance at 450 nm in the different concentrations of AFP. Half-maximal inhibitory concentration (IC50) which is the concentration of the 50% inhibitor, was used to evaluate the sensitivity of the antibody, and the lower IC 50 represents the higher the sensitivity of the antibody.28-30 The IC50 values can be obtained from the standard curves, IC50 value in 3D ELISA was 2.5 ng/mL, while in conventional ELISA it was 17.5 ng/mL, indicating that the antibody hybrid nanoflower exhibited higher sensitivity. Another factor to evaluate the sensitivity of the proposed method is the limit of detection (LOD). The value of LOD can be calculated according to the method described by Demchenmko: LOD = 3SB/S, SB represents the value of the standard deviation of blank samples, and S represents the slope of standard curve within the low concentration range. The nanoflower based 3D ELISA immunosensor exhibited an excellent detection performance of AFP with a linear range of 0.1-10 ng/mL (R2=0.990) (Figure 3e and f), and the LOD reached as low as 0.029 ng/mL (41.5 fM) (as illustrated in the Calculation in Supporting Information). Besides, in comparison, for conventional ELISA (2D ELISA), a linear relationship was plotted between A450 and the AFP concentration in the range of 5.0-50 ng/mL (R2=0.994) (Figure 3c and d), and the LOD was calculated to be 0.92 ng/mL. From this point, the sensitivity of the developed 3D immunosensor has been improved significantly compared with conventional 2D ELISA.

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The specificity of the nanoflower based 3D ELISA was evaluated by replacing the target AFP with BSA, SA, MUC-1, and CEA. We also explored the cross-reactivity of nanoflower based 3D ELISA targeting AFP in the presence of some nontarget homologous interfering proteins (MUC-1 and CEA). As depicted in Figure 4, obvious absorbances were observed in the presence of target AFP and mixed solution (AFP, MUC-1 and CEA), whereas BSA, SA, MUC-1, and CEA led to slight increase of absorbances. It can be concluded that the high specific binding of the antigen and antibody ensures the specificity of 3D immunosensing platform.

Nanoflower based 3D immunosensing platform for protein biomarker AFP analysis in human serum To evaluate reliability of the proposed immunoassay in the real samples, recovery test of AFP was performed using human serum samples as a real matrix. Four concentrations of AFP within the respective range were added into 10-fold diluted human serum samples. The recoveries of AFP detected in the serum samples ranges from 93.0% to 109.6% (Table 1). These results indicate the possibility of its application in real samples analysis.

Nanoflower based 3D immunosensing platform for EV71 determination The proposed nanoflower based 3D immunosensor as a pathogen detection platform was evaluated by employing ten-fold serial concentration of EV71 (7.396*103 – 7.396*109 copies/mL) in a standard buffer. Similarly, by changing the corresponding anti-EV71 antibody, the nanoflower based 3D immunosensing platform exhibited great versatility for EV71 determination as well (Scheme 1b). As shown in Figure 5 a and 5, the proposed 3D immunosensor showed a highly sensitive response to the target EV71 with the limit of detection as low as 7.396*103 copies/mL. However, for conventional HRP-based ELISA without the nanoflower based 3D platform, the lowest detectable concentration of EV71 is about 108 copies/mL,31 which indicates that our 3D platform exhibited great improvement in sensitivity with 105 times lower 12


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LOD.

Besides, compared with other reported method for EV71 detection, our

method offers satisfactory performance with much wider detection range (Table S1), from 7.396*103–109 copies/mL. These results further proved that 3D structure of the proposed immunosensor contributes a lot to the improvement of the sensitivity and detection range.

Nanoflower based 3D immunosensing platform for clinical sample analysis To further assess the 10 clinical positive samples of hand, foot and mouth disease (HFMD) patients that suffered from EV71 infection and 1 negative sample were tested by the proposed nanoflower based 3D immunosensor. All the EV71 clinical samples were diluted 5 folds without any other pretreatment. In the meanwhile, all these clinical samples had been diagnosed by using RT-PCR (Figure 5c), which is a widely used method in clinical diagnosis of HFMD. As shown in Figure 5d, the nanoflower based 3D ELISA achieved a complete consistency with the outcomes of RT-PCR, indicating the promising application in clinical sample analysis.

CONCLUSIONS Taking the advantage of huge loading capacity and the unique three-dimensional structure of hybrid nanoflower, we constructed a robust and versatile nanoflower based 3D immunosensing platform. This platform can be applicable to the detection of various kinds of targets by changing the corresponding antibody. For the determination of protein biomarker AFP as well as EV 71, the nanoflower based 3D immunosensing improves the sensitivity with a detection of limit of 0.029 ng/mL and ~103 copies/mL for AFP and EV71, respectively. For real human serum analysis, the 3D immunosensing platform performed an acceptable level of accuracy (recovery value between 93.0 and 109.6%). In clinical sample analysis, the method exhibited a complete consistency with RT-PCR result. In conclusion, the rapid and sensitive immunosensing platform provides a new choice for detection of various kinds of target and has promising potential in the early diagnosis of clinical samples. 13



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ASSOCIATED CONTENT Supporting Information Detailed

experimental

procedure,

calculation

of

encapsulation

efficiency,

determination of LOD, schematic representation of approaching process in 3D ELISA, the optimization of reaction condition, comparison of the reported sensor for AFP and EV71 detection (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Tel: +86 27-68756557. ORCID Zhike He: 0000-0002-0082-4056 Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGEMENTS This work was supported by National Major Science and Technology Projects (2018ZX10301405) and the National Natural Science Foundation of China (21475101, 21675119).

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Scheme 1. (a) Schematic synthesis of biotinylated capture antibody nanoflower; (b) Schematic illustration of nanoflower based 3D immunosensing platform for AFP and EV71. (the plate was blocked with BSA in the initial step; all the binding steps described below were followed by a rinsing step.)

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Figure 1. (a) SEM images of antibody (biotin)- Cu3(PO4)2 hybrid nanoflowers; (b) High-resolution SEM image of single antibody (biotin)- Cu3(PO4)2 hybrid nanoflowers (inset: the flower in nature); (c) High-resolution TEM image of single nanopetal in antibody (biotin) hybrid nanoflowers; (d) XRD patterns of hybrid nanoflowers obtained with antibody (red line), particles of crystals obtained without protein (black line), and standard Cu3(PO4)2•3H2O (JPSCD PDF#22-0548) (black inset); (e) Element mapping of five main elements in the hybrid nanoflowers, including oxygen, carbon , nitrogen, copper, and phosphorus, respectively; (f) EDS spectrum of complete element composition.

Figure 2. Storage stability of antibody hybrid nanoflowers: (a) The relative activity of hybrid nanoflower and free antibody in PBS (pH 7.4) at room temperature; (b) SEM images of the nanoflowers stored in PBS for two months.

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Figure 3. Representative Photographs taken from the (a) conventional colorimetric immunoassay and (b) nanoflower based 3D immunosensing platform of AFP standards, respectively; (c) The UV-vis absorbance spectra of conventional colorimetric immunoassay at different concentrations of AFP: 0, 5.0, 10, 20, 30, 40, 50, 60 ng/mL; (d) The calibration curve of conventional colorimetric immunoassay for the determination of the AFP concentration in PBS buffer (pH7.4); (e) The UV-vis absorbance spectra of nanoflower based 3D immunosensing platform at different concentrations of AFP: 0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, 25, 50, 75, 100 ng/mL; (f) The calibration curve of nanoflower based 3D immunosensing platform for the determination of the AFP concentration in PBS buffer (pH7.4).

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Figure 4. Speciﬁcity of the assay for AFP detection with different nontarget protein. Experimental conditions: 0.02 mg/mL antibody nanoflower, 10 ng/mL different protein, 0.02 mg/mL Ab.-HRP.

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Figure 5. (a)The UV-vis absorbance spectra of nanoflower based 3D immunosensing platform at different concentrations of EV 71: 7.396*103 –109 copies/mL (ten-fold dilution); (b) the calibration curve of nanoflower based 3D immunosensing platform for the determination of the EV71 in PBS buffer (pH7.4); (c) RT-PCR analysis of multiple clinical samples; (d) detection of multiple clinical samples by the proposed nanoflower based 3D immunosensing platform.

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Table 1. Recovery of AFP in healthy human serum in nanoflower based 3D ELISA.

Sample

Added

Found

Recovery

RSD (%)

number

(ng/mL)

(ng/mL)

1

0.5

0.53±0.02

106.0

6.8

2

1.0

0.93±0.73

93.0

2.7

3

5.0

5.21±0.05

104.2

3.6

4

10.0

10.93±0.47

109.3

1.3

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(n=3)

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Table of Contents

Nanoflower based 3D immunosensing platform with high capture efficiency was developed for sensitive determination of protein biomarker and Enterovirus 71.

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Three-Dimensional Immunosensing Platform Based on a Hybrid

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