Superwettable microchips with improved spot homogeneity toward sensitive biosensing

Superwettable microchips with improved spot homogeneity toward sensitive biosensing

Biosensors and Bioelectronics 102 (2018) 418–424 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsev...

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Biosensors and Bioelectronics 102 (2018) 418–424

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Superwettable microchips with improved spot homogeneity toward sensitive biosensing ⁎

T ⁎

Yanxia Chena, Li-Ping Xua, , Jingxin Mengb, Shaohui Dengc, Lulin Mac, Shudong Zhangc, , ⁎ Xueji Zhanga, , Shutao Wangb a Research Center for Bioengineering and Sensing Technology, Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, PR China b CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, CAS Center for Excellence in Nanoscience, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China c Department of Urology, Peking University Third Hospital, Beijing 100191, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Superhydrophilic Superhydrophobic Superwettable microchip Spot homogeneity Biomarker Biosensing

The high-quality spots in microchips are prerequisites for sensitive and accurate detection of biomarkers. In this work, the superwettable micropattern was constructed by introducing superhydrophilic microwells onto a superhydrophobic substrate. The sample can distribute homogeneously within the well-designed superhydrophilic microwells after droplet evaporation and form homogeneous deposit spots, which can be ascribed to the enhanced Marangoni effect in superwettable micropattern and the suppressed outward flow by 3D nanodendritic silica structure. Based on the improved homogeneity of spots, sensitive and accurate fluorescence readout could be obtained. The free prostate-specific antigen (f-PSA) microchip based on the superwettable micropattern was developed. This superwettable f-PSA microchip exhibits high sensitivity, excellent specificity and long-term stability, and a limit of detection as low as 10 fg mL−1 is achieved. Moreover, the superwettable f-PSA microchip can accurately detect human serum samples with excellent correlations with chemiluminescence immunoassay in the clinic, demonstrating its great potential as a sensitive and reliable sensing platform for biological analysis and clinical diagnosis.

1. Introduction Biomarkers, which have been shown to have signatures related to tumor classification, diagnosis, and disease progression, become more and more important in clinical medicine (Ludwig and Weinstein, 2005; Wu and Qu, 2015). Microchip is a popular research and screening tool for sensitive and accurate detection of biomarkers including circulating tumor cells (Lin et al., 2014; Nagrath et al., 2007; Pallela et al., 2016), nucleic acids (Jia et al., 2016; Lee and Jung, 2011; Sassolas et al., 2008; Xu et al., 2015), proteins (Arya et al., 2017; Chen et al., 2008; Hu et al., 2011; Nam et al., 2003) and exosomes (Vaidyanathan et al., 2014; Wei et al., 2013; Zhao et al., 2016). Microarray biochips with simple and convenient technology hold great promise as early diagnostic and prognostic tools. Importantly, the performance of biochips relies on the quality of microarrays, especially the homogeneity of spots, which will improve the reliability of biochips and expand their extensive application (Kudina et al., 2016; Li et al., 2016; Rickman et al., 2003). However, a common phenomenon of ring-like morphology (Deegan et al., 1997, 2000) has always been observed on substrate, which may ⁎

cause inhomogeneous signal and even lead to inaccurate and unreliable readout (Dugas et al., 2005; Moran Mirabal et al., 2007). Therefore, it is an important issue to control the spot homogeneity at the sensing interface. Many efforts have been devoted to improve the spot homogeneity including reducing the droplet scale (Shen et al., 2010), decreasing the evaporation rate by the humidity control (Fukuda et al., 2013; Mujawar et al., 2014) and inducing surface-tension gradient by surfactant addition (Diehl et al., 2001; Dugas et al., 2005; Rickman et al., 2003). In addition, introducing anisotropic particles (Yunker et al., 2011), surface functionalization (Kusnezow and Hoheisel, 2003; Mujawar et al., 2013) and fabricating three-dimensional (3D) structures (Ressine et al., 2003, 2005) also can improve the spot homogeneity. However, several factors may affect the spot homogeneity, such as biomolecule-biomaterial interaction and three-phase (solid-liquid-gas) interaction (Jonkheijm et al., 2008; Li et al., 2013; Wong et al., 2009). Thus, the challenge remains in developing a simple, economical and effective method for improving the spot homogeneity of biomolecules to meet the high standards of microarray.

Corresponding author. E-mail addresses: [email protected] (L.-P. Xu), [email protected] (S. Zhang), [email protected] (X. Zhang).

https://doi.org/10.1016/j.bios.2017.11.036 Received 6 July 2017; Received in revised form 26 October 2017; Accepted 9 November 2017 Available online 21 November 2017 0956-5663/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. (a) Schematic illustration of homogeneous spot deposition within superwettable micropattern after droplet evaporation. (b) SEM (top-view, sideview) and TEM images of the nanodendritic silica (SiO2) coating (from left to right). (c) Water contact angles of microwall (i) and microwell substrate (ii). (d) The microarray of methylene blue trihydrate (MBT) droplets in superwettable micropattern.

In this paper, we introduce superwettable micropattern as a facile platform to improve spot homogeneity without the need for additional additives. The superwettable micropattern is constructed by patterning of superhydrophobic substrate with superhydrophilic microwell arrays. Droplet containing analytes is pinned to the superhydrophilic microwell, while the superhydrophobic substrate can limit the spread of droplet like a microwall. Benefiting from the enhanced Marangoni effect in superwettable micropattern and the suppressed outward flow due to the high hydrodynamic flow resistance of 3D nanodendritic silica structure, the analytes can distribute homogeneously within the region of superhydrophilic microwell and homogeneous deposit spot was obtained after droplet evaporation (Fig. 1a). This work is important in unraveling those interfacial phenomena including the droplet evaporation and spot deposition at solid interface. Moreover, the free prostate-specific antigen (f-PSA) microchip based on the superwettable micropattern exhibits high sensitivity, excellent specificity and longterm stability, and a limit of detection as low as 10 fg mL−1 is achieved. The superwettable micropattern is promising as a simple and sensitive biosensing platform in clinical diagnosis and prognosis.

microscopy (Nikon, Ti-E, Japan). The optical images were captured by digital camera 60D (Canon Co., Ltd). Tetraethyl orthosilicate (98%, TEOS), n-octadecyltrichlorosilane (95%, OTS), (3-mercaptopropyl) trimethoxysilane (95%, MPTES), N-ymaleimidobutyryloxy succinimide ester (≥ 98%, GMBS), fluorescein isothiocyanate (≥ 90%, FITC), methylene blue trihydrate (MBT) were purchased from Sigma-Aldrich Co. Streptavidin (SA) was purchased from Amresco Commercial Finance, LLC. Human free prostate-specific antigen (f-PSA), two mouse antihuman f-PSA monoclonal antibodies (biotinylated capture antibody, Ab1-biotin; and FITC-labeled antibody, Ab2-FITC) were purchased from Shanghai Linc-Bio Science Co. LTD. Bovine serum albumin (BSA), human serum albumins (HSA), and carcinoembryonic antigen (CEA) were from R&D systems. Citrate buffer (10 mM, pH 6.0), the phosphate-buffered saline (PBS, 10 mM, pH 7.4), Tris-HCl buffer (10 mM, pH 8.2), Na2CO3-NaHCO3 buffer (10 mM, pH 9.6) were purchased from Sigma-Aldrich Co. Milli-Q water (Millipore Corp, 18.2 MΩ cm) was used in all the experiments.

2. Materials and methods

The superwettable micropattern was fabricated according to the followed procedures (Deng et al., 2011; Xu et al., 2015; Yang et al., 2014): (і) candle soot layer was deposited on clean glass slide (5 cm) at a constant speed (5 cm s−1) with deposition time of 10 s, (іі) silica shell was fabricated on the candle soot-coated substrate via chemical vapor deposition of TEOS (2 mL), catalyzed by ammonia solution (2 mL, 98%) in a desiccator, at 37 °C for 24 h, (ііі) the carbon@silica core-shell nanocomposite was calcinated for removing the carbon core at 550 °C for 1.5 h, (іv) after cooling down to room temperature, the nanodendritic silica substrate was immediately immersed into an OTS solution (1% OTS in anhydrous toluene) for 10 min, and rinsed with toluene and ethanol, dried with nitrogen gas and followed by heating solidification at 120 °C for 20 min, (v) the OTS-modified nanodendritic silica substrate was irradiated by a high-pressure mercury lamp UV (about

2.2. Fabrication of superwettable micropattern

2.1. Apparatus and reagents The morphologies of the nanodendritic silica (SiO2) were characterized by a field-emission scanning electron microscopy (SEM, JSM7500F, Japan) at the acceleration voltage of 5 kV after sputter-coating with gold. Transmission electron microscopy (TEM) image was performed with a JEOL JEM-2100F (JEOL, Japan) in 200 kV. Contact angle images were captured by OCA20 device (Dataphysics, Germany) with 2 μL water droplet (Milli-Q, 18.2 MΩ cm) at ambient temperature. Surface functional modifications were characterized by X-ray photoelectron spectroscopy (XPS, Thermo Scientific Escalab 250Xi). The fluorescence images were obtained using an inverted fluorescence 419

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Fig. 2. The spot morphologies on three substrates after droplets evaporation (from left to right: the hydrophilic glass, the hydrophobic glass and the superwettable micropattern (microwell size of 1.01 ± 0.01 mm)). (a) Water contact angles of different substrates. (b) Illustration of three-phase boundary area of droplets before and after evaporation on different substrates. (c) The optical images of methylene blue trihydrate (MBT) solution before and after evaporation on different substrates (the scale bar is 1 mm). (d) The intensity analysis of deposit spots on different substrates.

150 mW cm−2) through photomask for 30 min to form micropatterns with different wettability. Then the nanodendritic silica substrate (SiO2), OTS-modified SiO2 substrate (OTS-SiO2) and UV-irradiated OTSSiO2 substrate (UV-OTS-SiO2) were further characterized by XPS, respectively. The non-irradiated region remained superhydrophobic; in contrast, the UV-irradiated regions became superhydrophilic due to the photodecomposition of OTS. Thus, the superwettable micropattern was constructed (Fig. S1).

S3). Based on the optimal parameters, 2 μL f-PSA solution and 2 μL Ab2FITC solution were simultaneously drop-casted and incubated in an Ab1-immobilized microwell in 80% RH for 30 min. After immunoreaction, the superwettable microchips were rinsed with PBS, DI water, and dried. The results of the fluorescence detection were obtained by using an inverted fluorescence microscopy. Clinical samples of serum f-PSA from prostate cancer patients were also analyzed by using the same proposed superwettable microchips. All solutions were fresh prepared and all detection were carried out at room temperature.

2.3. Assay of f-PSA 3. Results and discussion First, the fresh prepared superhydrophilic microwells were modified with 4% (v/v) MPTES in ethanol for 1 h, and then treated with 0.25 mM GMBS solution in DMSO for 30 min, leading to the successful attachment of GMBS in the microwells. Next, a drop of 2 μL SA solution was drop-casted in each microwell and placed in 80% RH for 30 min. Then the microchip was rinsed with PBS to remove excess SA (Wang et al., 2011). The SA-immobilized substrate was characterized with XPS (For details see Supplementary materials, Fig. S2b and S2c). After that, a drop of 2 μL Ab1-biotin was spotted in SA-immobilized microwell and incubated in 80% RH for 30 min. After being rinsed with PBS (10 mM, pH 7.4), the Ab1-immobilized microwells were blocked with 1 wt% BSA solution in PBS for 30 min, and then followed by PBS rinsing and drying (Fig. S2). The parameters for detection of f-PSA were optimized including exposure time, incubation time, pH and the concentration of SA, Ab1biotin and Ab2-FITC (see the Supplementary material for details, Fig.

3.1. Characterization of superwettable micropattern The morphologies of the obtained substrate of superwettable micropattern were studied. The SEM images reveal that the substrate consists of SiO2 nanoparticles, forming a loose, nanodendritic network coating (Fig. 1b, left) with the thickness of 26.6 ± 0.3 µm (Fig. 1b, middle). The TEM image indicates the hollow shell structure of the SiO2 nanoparticles due to the removal of the carbon core (Fig. 1b, right). The OTS-modified nanodendritic SiO2-shell coating with low surface energy and porous nanostructure has high water contact angles of 157.5 ± 1.8° (Fig. 1c, i), indicating the superhydrophobicity of the surface. The superhydrophobic surface can limit the spread of water droplet like a microwall. After the degradation of OTS via UV irradiation through photomask, the exposed region was formed superhydrophilic microwell with a contact angle of around 0° (Fig. 1c, ii). Moreover, the 420

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and MBT molecules precipitated along the periphery before the contact line receding (Li et al., 2013). Thus the ring-like spot formed on this hydrophobic glass. As for superwettable micropattern, the evaporation induced Marangoni effect leads to a recirculating flow, which also coexists with outward flow (Garcia-Cordero and Fan, 2017; Hernandez-Perez et al., 2016). The magnitude of Marangoni effect is related with the droplet height. The taller the droplet is, the faster the velocity of the recirculating flow (Hu and Larson, 2005). 2 μL MBT droplet anchored within superhydrophilic microwell and formed a regular spherical droplet with air/water/superhydrophilic microwell as the main contact line (right in Fig. 2b, upper part). The height of droplet is high because of the small contact area (Fig. S5c), and the Marangoni induced recirculating flow was enhanced. Moreover, the concentrated MBT solution layer penetrated into the porous nanodendritic SiO2-shell coating of superhydrophilic microwell via capillary forces (right in Fig. 2b, lower part). The high hydrodynamic flow resistance to the spot edge of the 3D structure of nanodendritic SiO2-shell layer suppressed outward flow (Ressine et al., 2005). Thus the recirculating flow predominate over the outward capillary flow and the coffee ring effect was suppressed, which carry MBT molecules to the center of the droplet, producing a significant homogeneous deposit spot.

nanodendritic silica substrate (SiO2), OTS-modified SiO2 substrate (OTS-SiO2) and UV-irradiated OTS-SiO2 substrate (UV-OTS-SiO2) were further characterized by XPS, respectively. As shown in Figs. S1b and S1c, after the immobilization of OTS on SiO2 surface, a dramatic increase of C 1s signal was observed for the OTS-SiO2 substrate due to the presence of alkyl chain in OTS and the signal of Si 2p peak declined, suggesting that the SiO2 surface was covered with OTS. After UV-irradiating, a significant reduction of C 1s peak was observed, indicating the adequate degradation of OTS. Thus, the superwettable micropattern was constructed. Benefiting from the synergistic effect of superhydrophilic microwell and superhydrophobic microwall, the droplets of methylene blue trihydrate (MBT) aqueous solution could be steadily anchored in the superwettable micropattern and form a regular array without cross contamination (Fig. 1d). Thus, we fabricated the substrate of superwettable micropattern with nanodendritic SiO2-shell structures and we then further explored its performance. 3.2. Sample distribution on superwettable micropattern The sample distribution on superwettable micropattern after evaporation was investigated and compared with the planar substrates of the hydrophilic glass and the hydrophobic glass. As shown in Fig. 2 (left), the hydrophilic glass (clean glass slide without modification) has a water contact angle of 19.8 ± 3.8°. The droplet of methylene blue trihydrate (MBT) solution tended to spread over a larger area and form an irregular shape on the hydrophilic glass. After evaporation, most MBT molecules concentrated along the periphery of the solution droplet (Fig. S4a). The deposit spot (diameter of 3.68 ± 0.17 mm) displayed the same irregular shape with the initial droplets and obvious coffeering spot was formed with stronger intensity than the interior of spot. For the hydrophobic glass (middle of Fig. 2, OTS-modified glass slide) with water contact angle of 112.6 ± 3.2°, the MBT droplet was more regular and smaller than on the hydrophilic glass. After evaporation, obvious coffee-ring spot (diameter of 1.48 ± 0.06 mm) was also formed with uneven intensity between ring and interior (Fig. S4b). In comparison, for the superwettable micropattern (right in Fig. 2, Fig. S4c), the droplet was pinned in superhydrophilic microwell and form a regular spherical shape. Moreover, the distribution of MBT molecules in the well-designed superhydrophilic microwell is very homogeneous. The uniform intensity of the deposit spot (diameter of 1.03 ± 0.03 mm) further proved the superwettable micropattern was capable of improving the homogeneity of sample distribution. The morphology of MBT spot is highly correlated with substrate wettability. As shown in Fig. S5a, for the hydrophilic glass, the evaporation of MBT droplet follows the constant contact area mode. The contact line was pinned to the substrate, and the contact angle and drop height decreases as it dries out. The edges of the droplet evaporate at a higher rate than in the center, and solvent flow radially outward to the edges and carry MBT molecules to the contact line, thus leaving a distinct coffee-ring stain, which is consistent with previous results (Deegan et al., 1997, 2000). As for the hydrophobic substrate with low contact angle hysteresis (CAH, Δθ), the droplet evaporation is usually dominated by a constant contact angle and a receding contact line, and the condensed spots could be formed without distinct coffee-ring in several studies (Cui et al., 2012; Yang et al., 2015). Δθ is generally expressed in terms of the difference between the advancing angle (θa) and receding angle (θr), (Δθ = θa - θr) (Gao and McCarthy, 2006; McHale et al., 2004). However, in our study, the advancing and receding angle of MBT solution on this hydrophobic glass are θa = 120.0 ± 2.4°, θr = 67.5 ± 4.2°, respectively. Δθ of MBT solution on the hydrophobic glass is as large as 52.5° (Fig. S6). The strong CAH seriously affected the contact line withdrawing during the early stage of evaporation, and most of MBT molecules concentrated along the periphery of the droplet (Fig. S5b). When the contact angle decreased from 109.8° to 64.4° (1–3 in Fig. S5b), the contact line would be depinned, but the outward flow has carried most of MBT molecules to the edge

3.3. Fluorescence analysis of spot on superwettable micropattern The accuracy and sensitivity of signal readout is vital for the reliability of spot-based analysis. The fluorescence analysis of spot was investigated on three different substrates, respectively. 2 μL fluorescein isothiocyanate solution (FITC, 10−6 mol L−1) was drop-casted and dried on different substrates respectively. For the hydrophilic glass (Fig. 3a, left) and the hydrophobic glass (Fig. 3a, middle), FITC droplet tended to form coffee-ring spot. The uneven and weak fluorescence spot led to difficulty in accurate and sensitive signal readout. In comparison, the homogeneous fluorescence spot could be obtained within superwettable micropattern (Fig. 3a, right). Due to the improved spot homogeneity, sensitive and accurate fluorescence readout (23.1 ± 1.1) could be obtained by the superwettable micropattern. Furthermore, the fluorescence intensity is related with the diameter of superhydrophilic microwell. As shown in Fig. 3b, with the decreasing of microwell diameter from 523.6 ± 6.0 to 387.3 ± 5.1 µm, the fluorescent intensity increased because the enrichment effect of fluorescence molecules played a more important role. When the diameter of microwell was 387.3 ± 5.1 µm, the maximum fluorescence intensity was obtained. When the diameter of microwell further decreased to 190.8 ± 6.2 µm, the fluorescence intensity became lower due to the aggregation-caused quenching (ACQ) (Hong et al., 2011; Xu et al., 2015). In order to obtain high sensitivity of fluorescence assay, the optimal size of superhydrophilic microwell used in this study is 387.3 ± 5.1 µm. As a result, the superwettable micropattern with improved spot homogeneity and sensitivity have great potential as a biosensing platform toward biomarker detection. 3.4. Detection of f-PSA on superwettable microchips The superwettable micropattern offers a simple and effective approach to improve spot homogeneity, which is a prerequisite for fabricating reliable and sensitive biochips. As a proof-of-concept, f-PSA microchips based on superwettable micropattern were developed (Fig. 4a). In brief, after the biotinylated f-PSA antibody (Ab1-biotin) immobilized on the SA-immobilized microwells, f-PSA and FITC-labeled f-PSA antibody (Ab2-FITC) were added into Ab1-immobilized microwells. During the droplet evaporation accompanied by immunoreaction, sandwich-type structures were formed and finally homogeneous fluorescence spots were obtained for f-PSA detection. The fluorescence images (Fig. 4b, insert) indicate the homogeneity and repeatability of f-PSA sensing on the superwettable microchips. The 421

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Fig. 3. FITC aqueous droplet (2 μL, 10−6 mol L−1) was casted and dried on different substrates. (a) The fluorescence images and corresponding fluorescence intensities on the hydrophilic glass (left, a part of the periphery), the hydrophobic glass (middle) and in the superwettable micropattern (right, microwell size of 1.01 ± 0.01 mm). (b) The fluorescence images and corresponding fluorescence intensities in superhydrophilic microwells with different diameters.

Fig. 4. (a) Schematic illustration of f-PSA detecting on superwettable microchips. (b) The relationship between fluorescence intensity and f-PSA concentration based on the superwettable microchips. The inset is the fluorescence images (top left) of fPSA detection at various concentrations. (c) The specificity of the as-proposed superwettable microchips for f-PSA sensing.

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Table 1 Comparison of the superwettable microchips with clinical method for serum f-PSA detection. Serum f-PSA concentration (ng mL−1) serum sample No.

clinical diagnostic assay

superwettable microchips [mean ± SD, n=3]

Relative difference %

1 2 3 4 5 6 7 8 9 10

1.244 4.002 1.05 4.339 11.16 1.022 0.441 1.777 8.677 1.388

1.306 ± 0.488 3.926 ± 0.350 1.124 ± 0.161 4.446 ± 0.580 10.99 ± 1.287 1.053 ± 0.445 0.454 ± 0.124 1.855 ± 0.202 8.201 ± 0.387 1.395 ± 0.161

4.98 −1.90 7.05 2.47 −1.52 3.03 2.95 4.39 −5.49 0.50

fluorescence intensity gradually increased with the increasing of the concentration of f-PSA (0, 10–12, 10−10, 10−8 and 10−6 g mL−1). As shown in Fig. 4b, the fluorescence intensity increased linearly with the logarithm of f-PSA concentration from 10–12 to 10−6 g mL−1 with a calibration function of I = 20.4 log[c] + 332.2 (correlation coefficient of 0.998; I, the fluorescence intensity; c, the concentration of f-PSA). The detection of f-PSA at a concentration of 10 fg mL−1 was also achieved by this superwettable microchips and the signal/noise ratio is about 4 (Fig. S7a and S7b). In previous works, the limit of detection for f-PSA by other microarray-based methods are about nanogram or picogram levels (Brazhnik et al., 2015; Lang et al., 2014; Liu et al., 2016). Thus, the superwettable f-PSA microchips exhibited superior performance and achieved the sensitive detection of f-PSA. The specificity and stability of the superwettable microchips are of great significance in diagnostic analysis. To evaluate the specificity of the proposed superwettable microchips, several different interferes including BSA (10%), HSA (0.5×10−3 g mL−1) and CEA (0.5×10−3 g mL−1) were introduced in the detection of f-PSA (10−9 g mL−1). As shown in Fig. 4c, there were no remarkable changes in fluorescence intensity as compared with f-PSA alone. Moreover, when f-PSA was replaced by BSA (10%), HSA (10−3 g mL−1), and CEA (10−3 g mL−1) in the detection system, these fluorescence intensities were very low and comparable with the control (PBS without f-PSA). These results indicated the excellent specificity of the superwettable microchips for the detection of f-PSA. The stability of the superwettable microchips was also evaluated by the detection of f-PSA (10−9 g mL−1). There were no obvious changes of fluorescence intensity for superwettable microchips stored for 4 weeks (Fig. S7c), and the as-prepared superwettable microchips exhibited excellent stability.

4. Conclusions In conclusion, the superwettable micropattern with superhydrophilic microwells and superhydrophobic microwall was fabricated and introduced as a facile biosensing platform. Because of the enhanced Marangoni effect in superwettable micropattern and the suppressed outward flow due to the high hydrodynamic flow resistance of 3D nanodendritic silica structure, the highly homogeneous deposit spot and reliable signal readout could be achieved in superwettable micropattern. The f-PSA microchips based on the superwettable micropattern with improved spot homogeneity exhibited excellent sensitivity, specificity and stability. A low limit of detection of 10 fg mL−1 for f-PSA was realized based on the superwettable microchips. Moreover, the superwettable microchips achieved reliable clinical detection of serum f-PSA from prostate cancer patients, demonstrating that the as-designed superwettable microchips have great potential for clinical applications and may be a promising biosensing platform for early and accurate cancer diagnosis. Acknowledgments The work is supported by National Natural Science Foundation of China (21475009, 21475008, 21275017, 21501184, and 21425314), MOST (2013YQ190467), the Top-Notch Young Talents Program of China, Beijing Municipal Science & Technology Commission (Z131102002813058 and Z161100000116037) and Fundamental Research Funds of USTB (FRF-BY-16-015). Notes The authors declare no competing financial interest.

3.5. Analysis of clinical samples and evaluation of method Appendix A. Supplementary material The superwettable microchips with improved spot homogeneity exhibit excellent repeatability, sensitivity, specificity and stability for fPSA sensing. The as-prepared superwettable microchips may be used in the clinical detection. The reliability and applicability of the as-proposed superwettable microchips were investigated by detecting serum f-PSA from prostate cancer patients. The test results of clinical samples by the superwettable microchips were compared with the clinical method based on chemiluminescence. As shown in Table 1, the superwettable microchips demonstrated a deviation ranging from −5.49% to 7.05% with those obtained from clinical method. In short, these above experiment results indicated that the as-proposed superwettable microchips were comparable to the clinical assay, exhibiting great potential as a reliable platform for clinical detection of serum f-PSA. In the near future, the proposed superwettable platform will be combined with commercial slide spotting robots to achieve high-throughput biomarker detections.

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