Highly Sensitive and Automated SERS-based Immunoassay for H5N1

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Highly Sensitive and Automated SERS-based Immunoassay for H5N1 Detection with Digital Microfluidics Yang Wang, Qingyu Ruan, Zhi-Chao Lei, Shui-Chao Lin, Zhi Zhu, Leiji Zhou, and Chaoyong James Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00002 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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Analytical Chemistry

Highly Sensitive and Automated SERS-based Immunoassay for H5N1 Detection with Digital Microfluidics Yang Wang1#, Qingyu Ruan1#, Zhi-Chao Lei1, Shui-Chao Lin1, Zhi Zhu1, Leiji Zhou1 and Chaoyong Yang1,2,*

1: MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Engineering, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.

2: Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200240, China.

# These authors contributed equally to the manuscript.

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) * To whom correspondence should be addressed.

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Abstract: Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface Enhanced Raman Scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4MBA) were synthesized with a core@shell nanostructure, and showed strong signals, good uniformity and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected througth the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (< 1 h) and lower reagent consumption (~ 30 µL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Keywords: Digital Microfluidics, SERS, Immunoassay, H5N1

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Introduction Digital Microfluidics (DMF) is an emerging fluid-handling technology that manipulates fluids as discrete droplets on a substrate. By applying electrical potentials to an array of electrodes on the substrate, droplets can be individually driven to transport, merge, split, and dispense from reservoirs based on the principle of electrowetting on dielectric (EWOD).1,2 The DMF platform has been integrated with many other techniques, such as mass spectrometry3, optical techniques4,5, electrical techniques6, and nuclear magnetic resonance7,8. This flexibility, high reconfigurability and facile coupling with other techniques make DMF well-suited for a broad range of applications, like enzyme assays9, DNA-based applications10, and cell-based applications11, especially those requiring complex, repetitive and long multistep protocols, such as immunoassays2,12. DMF allows immunoassays to be performed with less incubation time and lower reagent consumption compared with macroscale methods, because diffusion distances are two orders of magnitude smaller.13 Binding kinetics during each step is also accelerated relative to rates in bulk solution, as the biomolecules spend less time in contact with the assay surface, coupled with the effective mixing. More importantly, repetitive washing and long multistep incubations are performed automatically, which saves manual labor resources and affords reproducible results. In addition, it is favorable to manipulate various infectious materials by DMF, as its automated operation avoids direct manual contact with these infectious materials. These distinct characteristics of DMF make it a popular platform for implementing miniaturized quantitative

and sensitive immunoassays for various

biomarkers. According to previous reports, the detection readouts of immunoassays on DMF have generally been based on chemiluminescence4,12,14, fluorescence2,15-18 and electrochemical detection.13,19 These analytical detection tools have been effectively applied on DMF with good performance, but some limitations have emerged. The utility of enzymes is generally associated with high detection cost and poor stability. The inherent limitation of fluorescence is photobleaching, which can result in poor reproducibility. The complex and intricate electrode fabrication protocol on DMF limits the development of the

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electrochemical detection methods. As a consequence, there is great need for a new simple, low-cost, and sensitive readout tool for immunoassays on DMF. Surface enhanced Raman scattering (SERS), an ultrasensitive vibrational spectroscopic technique that can provide fingerprint information for a single molecule, is considered to be a promising alternative in bioanalysis.20,21 SERS-based biosensors have been developed and widely used in DNA analysis22, protein detection23-25 and bioimaging26-28 due to SERS’ extraordinary enhancement effect, high spatial resolution, excellent photostability, powerful multiplex detection ability, and absence of interference from water.29,30 More recently, novel SERS-active nanoprobes with the core-molecule-shell-nanostructure have been reported with large numbers of uniform “hot spots” in a narrow gap (sub nm to nm) to produce strong and reproducible SERS signals, engendering a large variety of SERS-based applications.29-33 In this work, we propose integrating DMF with SERS (DMF-SERS) for automated and sensitive bioanalysis. A SERS tag is designed with nanometer-sized gold core and silver shell and embedded with 4-mercaptobenzoic acid (4-MBA) molecules as Raman reporters and framework components. Therefore, SERS tags with strong signals, good uniformity and high stability could be obtained, which is crucial for quantitative analysis. Silver is chosen for coating because the enhancement ability of silver is much stronger than that of gold substrates.34 A sandwich immunoassay is designed where magnetic beads coated with antibodies are used as solid support to capture antigens from samples and form a beadsantibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. We also designed a highly sensitive and automated SERS-based immunoassay for H5N1 with DMF. The automation capability of DMF can greatly simplify the assay procedure, while reducing the risk of contamination from various hazardous samples. In addition, the small reaction volume allows short assay times and low reagent consumption. Furthermore, the integration of the SERS technique, converting the molecular recognition into amplified SERS signals, greatly improves the assay performance. Therefore, this DMF-SERS method holds great potential for automated and sensitive detection of a variety of infectious diseases. 4


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Experimental Reagents and Materials. Dynabeads® M-280 Tosylactivated and Streptavidin-HRP were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Mouse anti-H5N1 hemagglutinin monoclonal antibody, biotinylated rabbit anti-H5N1 hemagglutinin polyclonal antibody and recombinant H5N1 hemagglutinin were purchased from Sino Biological Inc. (Beijing, China). Biotin-PEG-SH (MW~5 kDa) was obtained from JenKem Technology Co., Ltd. (Beijing, China). SU-8 (GM 1040) was provided by Gersteltec Sarl (Switzerland).

4-Mercaptobenzoic

acid

(4-MBA)

was

purchased

from

Aladdin

Biochemical Technology Co., Ltd (Shanghai, China). HRP and Pluronic F127 were from Sigma-Aldrich (St. Louis, MO, USA). Tris was obtained from Sangon Biotech Co., Ltd (Shanghai, China). Chloroauric acid hydrate (HAuCl4·4H2O), sodium citrate, silver nitrate (AgNO3), and sodium chloride (NaCl) were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Unless stated otherwise, all chemicals and materials were analytical grade. The buffer solutions used were as follow: TBS (20mM Tris, 150mM NaCl, pH 7.4); wash buffer (TBS with 0.1 wt % pluronic F127); sample dilution buffer (TBS with 0.1 wt % BSA and 0.1 wt % pluronic F127); detection antibody dilution buffer (TBS with 0.5 wt % BSA and 0.1 wt % pluronic F127); buffer A (0.1 M Na-phosphate, pH 7.4); buffer B (PBS with 0.1% (w/v) BSA, pH 7.4); coupling buffer (0.1 M Na-phosphate with 3 M ammonium sulphate, pH 7.4); blocking buffer (PBS with 0.5% (w/v) BSA, pH 7.4); stop solution (2 M H2SO4). Sample dilution and detection antibody dilution buffers were filtered with 0.2 µm filters before use. Chip design and fabrication. The DMF chip, designed by AutoCAD software, consists of two parallel glass plates (Figure 1c). The bottom plate was coated with 300 nm thick chromium and patterned through photolithography and etched to form an electrode array. As shown in Figure 1a, the electrode array consists of 30 actuation electrodes (2.2 mm × 2.2 mm each) and 6 reservoir electrodes (5.4 mm × 3.6 mm each), with inter-electrode gaps of 20 µm. Then the bottom plate was coated with SU-8 1040 photoresist (~11.7 µm) as a dieletric layer to insulate electrodes and droplets. The top plate has an indium tin oxide (ITO) coating to serve as a ground electrode. All surfaces of the two plates were 5



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hydrophobized with Teflon AF 1600 (~200 nm). The two plates were separated by ~360 µm thick double-sided tape (Scotch Brand), and filled with fluorocarbon oil (FC 40). The volume of the unit actuation electrode and reservoir electrode droplet were 1.7 µL and 7 µL, respectively. EWOD system. The automated platform consists of a DMF driving system (function generator), Microfluidic software, DMF chip, webcam, magnet and an integrated Raman system. A photo of DMF-SERS platform is displayed as Figure S1. The chip was positioned on the chip holder, which was connected to the DMF driving device via a Pogo pin interface (48 pins). Droplet movement was driven by applying sine wave potentials (90~120 Vrms, 10 kHz) between the top plate (ground electrode) and electrodes on the bottom plate. Droplet manipulation, controlled by software, was monitored and recorded by a webcam. Reagents were loaded into the chip by pipetting droplets into the gap between the two plates. When applying driving potentials to the adjacent reservoir electrodes, droplets could be introduced into the chip.14 Waste and unused reservoir droplets were moved away by filter paper. Unit droplets were dispensed from reservoirs by actuating successive adjacent electrodes on the bottom. To implement a mixing operation, a program was set to allow droplet shuttle across four electrodes in a circle. Bead separation was achieved by positioning a magnet underneath the chip, and the supernatant was moved to the waste reservoir. When a aqueous droplet contains proteins, its motion become difficult and eventually immovable, as proteins tend to accumulate on the chip surface, the hydrophobic surface becomes hydrophilic, resulting in shortening the chip lifetime.35 To prevent this “biofouling” effect on the surface of the DMF chip, non-ionic polymer surfactant Pluronic F127 was added to buffers to reduce the degree of protein adsorption and aid in droplet movement.2,35 Synthesis of SERS tags. The SERS tags were fabricated with a core-shell nanostructure and embedded with 4-MBA (50 nm Au@MBA@Ag, 5 nm).36 Figure S2 illustrates the synthetic scheme of SERS tags. First, AuNPs (Figure S3) with an average diameter of 50 nm were synthesized by the reduction of HAuCl4 with sodium citrate (More detail in 6


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supporting information).37 Then AuNPs were functionalized with 4-MBA molecules. These molecules adsorbed to the surface of AuNPs through Au-S bonds.38 4-MBA (1.0 mM dissolved in ethanol) was slowly added to the colloidal gold to a final concentration of 3 µM with gentle stirring in a round-bottom flask at room temperature for 1 h. After incubation, the mixture was centrifuged at 5000 rpm for 5 min to remove free 4-MBA. The fuscous gold sediment was resuspended with freshly prepared 27 µM sodium citrate to avoid AuNP aggregation, because the citrate layer can stablize nanoparticles due to the electrostatic repulsion.39 Next, the resuspended gold nanoparticles (46 pM, 5 mL) were boiled with stirring and refluxing. Afterward, AgNO3 (20 mM, 100 µL) and sodium citrate (20 mM, 300 µL) were added successively drop-by-drop with vigorous stirring. Finally, 4MBA-labeled SERS tags were obtained after boiling for 20 min. For use in immunoassays, SERS tags were functionalized with biotin. Biotin-PEG-SH (100 µM, 47 µL) was added to the freshly prepared SERS tags (46 pM, 1 mL) and incubated at room temperature for 1 h. Thereafter, free biotin-PEG-SH was removed by centrifugation (5000 rpm, 5 min) and resuspension. The synthesized SERS tags could be preserved at room temperature for a long time with high stability. Preparation of capture antibody-coated magnetic beads. Dynabeads® M-280 Tosylactivated beads (165 µL, 30 mg/mL) were washed three times with buffer A. After removing the supernatant, H5N1 capture antibody (1 µg/µL, 100 µL) and coupling buffer (50 µL) were added and incubated at 37 °C for 12~18 h. After removing the suspernatant, blocking buffer (1 mL) was added and incubated at 37 °C for 1 h to block the uncoupled sites on the beads. Next, the blocked beads were washed three times and resuspended in buffer B (240 µL). Finally, the capture antibody-coated magnetic beads (CA-M-Beads) were obtained and stored at 4 °C (20 mg/mL). SEM was performed to characterize the morphology of CA-M-Beads (Figure S4). In order to further investigate the coupling of capture antibody on magnetic beads, a second antibody labeled with Alex 488 was used to stain CA-M-Beads. Figure S5 indicates that the capture antibody was successfully coupled on magnetic beads.

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Washing efficiency characterization. An essential factor of determining assay performance is the washing procedure. We evaluated the washing efficiency by the following method: CA-M-Beads were dispersed in 50 ng/mL free HRP at a final concentration of 5 mg/mL. A 2 µL mixing droplet was loaded on the chip, and covered with the top plate. The magnet was positioned underneath the chip to immobilize magnetic beads, and most of the supernatant was removed by actuating a series of adjacent electrodes. Then beads were washed with a wash buffer droplet (1.7 µL) which was dispensed from the reservoir. The washing supernatant was collected after each wash cycle using a pipette and transferred to 96-well plate respectively. The amount of HRP in the supernatant determined the washing efficiency. Substrate TMB (250 µL) was added to each well and incubated for 15 min. Absorbance was read at 450 nm using a plate reader after stop solution (50 µL) was added. The same experiment was performed in-tube with the same volume for comparison. Similarly, magnetic beads were enriched by a magnet, and the supernatant was collected by the pipette after each wash cycle. Detection antibody dilution buffer with the substrate was also measured on the same plate reader as the negative control. Procedure of DMF-SERS method. The SERS-based immunoassay was performed according to a standard sandwich ELISA protocol. Typically, a droplet of CA-M-Beads (2 µL, 5 mg/mL) was loaded into the chip and surrounded by fluorocarbon oil (FC 40). Other reagents (7 µL), including sample, biotinylated detection antibody, streptavidin, biotinylated SERS tags, and wash buffer were introduced into their respective reservoirs as described above. First, a unit sample droplet was dispensed from the reservoir and mixed with the preloaded CA-M-Beads droplet. The mixture was actively incubated for 5 min. After that, the beads were attracted by the magnet, and unreacted reagents were moved to the waste reservoir. After every incubation, the beads were washed three times. Then the beads were isolated and mixed with a detection antibody droplet in the same way, to form the immunocomplex on magnetic beads. Afterwards, the beads were incubated with streptavidin and SERS tag successively using the same procedure to obtain the SERS tagfunctionalized immunocomplex (Figure 1d). For the last incubation, the beads were

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washed for five times. After adequate washing, the SERS signal was then read with a portable iRaman Plus (Figure 1a). SERS measurements. SERS spectra were acquired from a portable iRaman Plus (B&W TEK, Shanghai, China) with a 785 nm excitation laser with power approximately 320 mW. The objective used was a 20 × long working distance lens (NA = 0.4) for focusing the laser beam into the sample and collecting and directing the scattered light to the charge-coupled device (CCD) detector. The spectra were collected from 500 to 3000 cm-1 with a spectral resolution of 3 cm-1. SERS tags were measured with 10 % power (~ 30 mW) and a single 10 s accumulation, while SERS measurements in immunoassays were performed using 100 % power (~ 300 mW). As shown in Figure 1b, two characteristic Raman peaks (1071 cm-1 and 1580 cm-1) of 4-MBA were obviously distinguished, and the 1071 cm-1 peak was used for quantitative analysis of the H5N1 antigen.

Results and Discussion Working principle of the DMF-SERS method. Figure 1 illustrates the working principle of the DMF-SERS method for automated and sensitive immunoassay. First, reagents are introduced into reservoirs of the chip using the method described in the experimental section. Then unit droplets of sample and detection antibody are dispensed from reservoirs to incubate with CA-M-Beads successively to form an immunocomplex. Efficient incubation and washing steps are realized automatically through DMF actuation. Afterwards, sequential incubation with strepavidin and biotinylated SERS tags can functionalize the complex beads with SERS tags. After adequate washing, the SERS signal is read with a portable iRaman Plus. As a result, antigen can be sensitively detected through the strong SERS signals. Quantitative analysis can be realized by measuring the Raman intensity of 4-MBA at the 1071 cm-1 peak, as the intensity at 1071 cm-1 is slightly stronger than that at 1580 cm-1. The intensity is correlated with the amount of SERS tags, which is determined by the target concentration.

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Figure 1. Schematic illustration of SERS-based immunoassay with digital microfluidics. (a) Illustration of DMFSERS method and bottom plate of DMF chip. (b) Two characteristic Raman peaks of 4-MBA at 1071 cm-1 and 1580 cm-1. (c) Side view of DMF chip containing a droplet with magnetic beads. (d) Immunocomplex functionalized with SERS tags on magnetic beads.

Working performance of DMF chip. Generally, the uniformity of the sample or reagent droplet volume would significantly affect the assay performance. Hence we first investigated the volume variation of droplets dispensed from reservoirs. The volume of droplet is determined by its area since the height of the space between two plates is fixed. Figure 2a and 2b show the areas of 20 dispensed droplets (~1.7 µL) from one reservoir and different reservoirs with CV(%) of 3.54% and 4.45%, respectively, which are comparable with that of 2.22% by manual pipetting (Figure S6). The results demonstrated good uniformity and reproducibility of droplets dispensed from reservoirs. Another essential factor for determining heterogeneous immunoassay performance is washing. The washing step on-chip is comprised of bead separation and resuspension processes. The magnetic beads are enriched by a permanent magnet underneath the chip, and the excess unreacted reagents are removed after each binding step. After that, the enriched magnetic beads are resuspended with a fresh wash droplet. The washing

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efficiency of DMF was further evaluated. Capture antibody-coated magnetic beads CA-MBeads were first dispersed in an HRP droplet and introduced into the chip. Washing was applied by immobilizing magnetic beads with the magnet, then most of the supernatant was removed and collected after each washing operation. The amount of HRP in the supernatant of each washing step was analyzed to evaluate the washing efficiency. This washing process was performed both on-chip and in-tube for a direct comparison with the same volume (1.7 µL) of washing droplet. As shown in Figure 2c, every point represents the end absorbance of supernatant collected on-chip (black) and in-tube (red). With increasing number of washes, the amount of HRP in the supernatant decreased dramatically, resulting in reduction of the absorbance. Furthermore, the absorbance reached the negative control level after three washes in both cases. With the same washing efficiency, the washing step realized by automated DMF chip can replace conventional wash steps for efficient washing.

Figure 2. Volume variation of dispensed droplets and washing efficiency characterization. (a) Area of 20 droplets dispensed from one reservoir with CV (%) of 3.54%. (b) Area of 20 droplets dispensed from different reservoirs with CV (%) of 4.45%. (c) Washing efficiency characterization on-chip and in-tube (The dashed line means negative control).

Synthesis and characterization of SERS tags. SERS tags were fabricated with core-shell nanostructure and embedded with 4-MBA as Raman reporter and framework molecules. Because these molecules are trapped between the core and shell, a narrow gap is formed with a large number of uniform “hot spots” for a significant SERS enhancement. Therefore strong, reproducible and uniform SERS signals are obtained for quantitative analysis. UV-Vis spectroscopy and transmission electron microscopy (TEM) were used to characterize SERS tags, and SERS spectra were also scanned to validate the SERS activity. As Figure 3a shows, the gold core

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showed a dark red appearance with a characteristic surface plasmon resonance (SPR) peak at 520 nm, and the color changed to orange along with the band shifted to around 420 nm after silver shell coating. Furthermore, TEM images (Figure 3b) of SERS tags demonstrated good uniformity and dispersity of these particles. Besides, the diameter (Figure 3c) of AuNPs and SERS tags were found to be around 49.47 ± 6.09 nm and 60.46 ± 3.35 nm, respectively. Moreover, STEM characterization and EDS (Energy Dispersive Spectrometer) were also performed using HRTEM (high-resolution transmission electron microscopy). As shown in Figure 3d, a light silver shell surrounds the bright gold core, demonstrating successful coating of silver shell. The irregular thickness of silver shell was results from silver atoms preferred depositing on silver shell rather than gold core.30,40,41 In addition, elemental mapping with EDS was performed to demonstrate the elemental distribution on SERS tags. As Figure 3d exhibits, Au, S, and Ag elements were marked with blue, red and yellow, respectively. The merged image reveals a distinct core-shell structure and a monolayer of 4-MBA molecules buried between the core-shell nanostructure. These results clearly indicated that SERS tags with perfect structure and good uniformity were obtained. After characterizing the structure of SESR tags, optimization was conducted to achieve greater enhancement. SERS tags were optimized from three factors: diameter of the gold core, amounts of 4-MBA, and thickness of the silver shell (More details in Supporting Information and Figure S7). It was concluded that 50 nm gold core, full monolayer amounts of 4-MBA and 5 nm thickness silver shell had the most enhancement and were chosen for SERS tags synthesis. In order to quantitatively evaluate the enhancement of SERS tags, SERS enhancement factor (EF) was calculated. The analytical enhancement factor (AEF) was calculated to be 6.71×105 (More details in Supporting Information and Figure S8).

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Figure 3. Characterization of SERS tags. (a) UV-Vis spectra (inset: images) of AuNPs and SERS tags. (b) TEM images of SERS tags with 5 nm thickness silver shells. (c) Diameter distributions of AuNPs (49.47 ± 6.09 nm) and SERS tags (60.46 ± 3.35 nm). Diameter was measured by Nanomeasurer software, and analyzed from 50 particles. (d) STEM image and Elemental mapping of SERS tags using HRTEM.

In order to realize the SERS labeling step in immunoassay, SERS tags are functionalized with biotin-PEG-SH, which is adsorbed on the surfaces of SERS particles through Ag-S bonds. Zeta potential (Figure S9) and TEM (Figure S10) were conducted to verify biotinPEG-SH was successfully functionalized on the surface of SERS tags. In addition, the activity of biotinylated SERS tags was measured to investigate whether biotin-PEG-SH molecules have effects on SERS tags. As Figure 4a exhibits, the SERS intensity was decreased about 28% after biotin functionalized. We speculated that these molecules with long-chain wrapped around SERS particles would affect the activity of SERS tags. Besides, the intensity decreased about 48% after washing, possibly due to partial loss of SERS particles during centrifugation and washing steps. Furthermore, the quantitative detection ability of SERS tags was tested. As Figure 4b shows, with increasing SERS tag concentration, the Raman intensity increased correspondingly, indicating that SERS tags with good uniformity and reproducibility could be applied for quantitative analysis. Finally, the stability of SERS tags and biotinylated SERS tags were investigated for two months. As shown in Figure 4c, Raman intensity of SERS tags dropped down dramatically, while that of the biotinylated SERS tags only

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decreased slightly after stored at room temperature over more than 60 days. It could be concluded that these particles are more stable after biotin functionalization, because biotinPEG-SH molecules wrapped around the particle surface can stabilize these particles.40 Therefore, SERS tags with strong activity, good uniformity and high stability were obtained and could be used for highly sensitive SERS-based immunoassay.

Figure 4. (a) Raman spectra of SERS tags and biotinylated SERS tags with two characteristic Raman peaks at 1071 cm-1 and 1580 cm-1. (b) Linear response of SERS signal at 1071 cm-1 to SERS tag concentration, suggesting good uniformity and reproducibility of the probe. (c) Stability of SERS tags and biotinylated SERS tags. The intensity of peak at 1071 cm-1 was used for measurement, and each point was measured three times.

DMF-SERS method for quantitative detection of H5N1. Using the DMF-SERS method, a sandwich assay was designed for H5N1 detection by functionalizing the immunocomplex with SERS tags. Figure S11 depicts the SERS-based immunoassay steps on-chip, involving reagent loading, immune reaction, SERS labeling, and Raman detection. Figure 5a shows the SERS spectra collected from sandwich immunocomplexes of samples with six different concentrations. The intensity of Raman spectra increased with increasing antigen concentrations. The peak at 1071 cm-1 was chosen for quantitative analysis. As shown in Figure 5b, the SERS intensity was linearly related to the concentration of H5N1, demonstrating its feasibility for H5N1quantitative detection. To investigate the selectivity of the DMF-SERS method for H5N1 detection, several common antigens were chosen with 10× molar concentration of H5N1 as negative targets, including prostate specific antigen (PSA), C-reactive protein (CRP), hepatitis B surface antigen (HBsAg), and cardiac troponin T (cTnT). As shown in Figure 5c, H5N1 exhibited a significant Raman signal, while the negative antigens showed only negligible signals. These results demonstrated that DMF-SERS method is highly selective for H5N1 analysis.

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Figure 5. (a) SERS spectra for increasing concentrations of H5N1 sample: (1) 0 pg/mL, (2) 500 pg/mL, (3) 1500 pg/mL, (4) 2500 pg/mL, (5) 4000 pg/mL, (6) 5000 pg/mL. (b) Calibration curve of DMF-SERS method for H5N1 detection in buffer. The error bars indicate standard deviations from three parallel experiments performed on-chip. (c) The selectivity of DMF-SERS method (H5N1: 0.085 nM; PSA, CRP, HbsAg: 0.85 nM; cTnT: 0.135 nM).

Real sample analysis. To demonstrate the feasibility of the DMF-SERS method for use in complex biological matrices, different concentrations of H5N1 were spiked into human serum for analysis. The serum droplet was difficult to move on the DMF chip, as it contained large amounts of proteins. Hence pluronic F127 (0.1 wt%) was added to human serum to improve the motion of the serum droplet. As shown in Figure 6a, the SERS intensity was linearly correlated with the H5N1 concentration in serum and a calibration curve was obtained with an LOD of 74 pg/mL, which is much lower than the LOD of the standard ELISA method of 399 pg/mL (Figure S12b), indicating good anti-interference ability and high sensitivity of this method in real sample analysis. The conventional ELISA method relying on enzyme-based coloration signal readout has a narrow optical change range, resulting in limited signal amplification ability. In contrast, SERS is an excellent vibrational spectroscopic technique with large enhancement factor (103 to 1010). Therefore, SERSbased detection method could reach an ultra-sensitivity level to detect a single molecule, which is extremely favorable for trace analysis.42 Therefore, SERS-based method greatly improves the assay sensitivity compared with the standard method. Furthermore, the accuracy of the DMFSERS method was evaluated by comparing with the standard ELISA method. For six sample tests, results from DMF-SERS were found to agree well with the standard ELISA method with a slope of 1.08 (Figure 6b), suggesting the strong correlation of two methods. Moreover, Bland-Altman analysis was conducted to verify the agreement between two methods. As Figure 6c displays, the DMF-SERS method had a bias offset of -46.16 at the

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95% confidence level (± 1.96 SD) from -336.1 to 243.8 for H5N1 detection compared to the standard method. These data demonstrated excellent accuracy of the DMF-SERS method. The standard ELISA method generally requires labor-intensive procedures with long time processing (6 h) and large reagent volume consumption (~ 650 µL), which greatly increases the assay cost. In contrast, the DMF-SERS method involves less assay time (~ 50 min) and lower reagent consumption (~ 30 µL). In addition, this method shows good accuracy and anti-interference ability in real sample analysis. Furthermore, automated operation can greatly simplify the assay procedure, while reducing the risk of exposure to hazardous samples. Moreover, multiple parallel assays could be realized on the DMF platform with an extended electrode system and integrated electromagnet. Therefore, the DMF-SERS method shows excellent performance and is highly applicable as a sensitive tool for detection of various diseases.

Figure 6. (a) Calibration curve of DMF-SERS method for H5N1 detection in human serum. (b) Correlation analysis between DMF-SERS method and standard ELISA method for H5N1 detection in six serum samples. (c) Bland-Altman analysis for the agreement between DMF-SERS method and standard ELISA method of six serum samples. Data shows a 95% confidence interval of the mean.

Conclusions In summary, a SERS-based immunoassay method with DMF was developed for rapid, automated and sensitive detection of influenza virus H5N1. To the best of our knowledge, this is the first report integrating a SERS-based immunoassay with DMF. Automated and quantitative analysis of avian influenza virus H5N1 was realized on DMF in buffer and human serum. Our DMF-SERS method presents several distinct advantages. The automation capability of DMF can greatly simplify the assay procedure, cut down assay

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time and reduce the risk of exposure to hazardous samples. Our method allows ultrasensitive detection of H5N1 within 1 h, which is 6-fold shorter than the time for conventional ELISA (6 h). Secondly, the DMF dramatically decreases the volume of sample and reagents. It requires only 30 µL total of reagent, as compared to 650 µL for ELISA. Low reagent consumption decreases assay cost. Furthermore, the integration of DMF with SERS greatly improves assay performance owing to the ultrasensitivity of SERS. A much lower LOD was achieved with DMF-SERS compared to that of a standard ELISA. Finally, because of the excellent spatial resolution of SERS and parallel liquid processing capability of DMF, the DMF-SERS method can be easily extended to multiplex analysis. Combining the automation capability of DMF and ultrasensitivity of SERS, the DMF-SERS platform is a new powerful tool for a broad range of applications in biological analysis.

Conflicts of interest All authors have declared that no competing interest exists.

Supporting Information A photo of DMF-SERS platform, synthesis of SERS tags, synthesis of 50 nm AuNPs, CAM-Beads characterization, variation of droplet volume by manual pipetting, SERS tags optimization, SERS enhancement factor calculation, SERS tags biotin functionalization, DMF-SERS method for H5N1 immunoassay, and feasibility of H5N1 immunoassay.

Acknowledgments We thank the National Natural Science Foundation of China (21735004, 21435004, 21775128,

21705024, 21521004), and Program for Changjiang Scholars and Innovative

Research Team in University (IRT13036) for their financial support.

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Highly Sensitive and Automated SERS-based Immunoassay for H5N1

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