Multiplexed Detection of Cytokines Based on Dual Bar-Code Strategy

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Multiplexed detection of cytokines based on dual bar-code strategy and single-molecule counting Wei Li, Wei Jiang, Shuang Dai, and Lei Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03043 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 8, 2016

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Analytical Chemistry

Multiplexed detection of cytokines based on dual bar-code strategy and single-molecule counting

Wei Li a, Wei Jiangb, Shuang Dai a, Lei Wanga, *

a

Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan, 250012, P. R. China.

b

School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, P. R. China.

Corresponding author: Tel: +86 531 88380036. Email: [email protected]

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ABSTRACT Cytokines play important roles in immune system and have been regarded as biomarkers. While single cytokine is not specific and accurate enough to meet the strict diagnosis in practice, in this work, we constructed a multiplexed detection method for cytokines based on dual bar-code strategy and single-molecule counting. Taking interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) as model analytes, firstly, the magnetic nanobead was functionalized with the second antibody and primary bar-code strands, forming a magnetic nanoprobe. Then, through the specific reaction of the second antibody and the antigen that fixed by the primary antibody, sandwich-type immunocomplex was formed on the substrate. Next, the primary bar-code strands as amplification units triggered multibranched hybridization chain reaction (mHCR), producing nicked double-stranded polymers with multiple branched arms, which were served as secondary bar-code strands. Finally, the secondary bar-code strands hybridized with the multimolecule labeled fluorescence probes, generating enhanced fluorescence signals. The numbers of fluorescence dots were counted one by one for quantification with epi-fluorescence microscope. By integrating the primary and secondary bar-code-based amplification strategy and the multimolecule labeled fluorescence probes, this method displayed an excellent sensitivity with the detection limits were both 5 fM. Unlike the typical bar-code assay that the bar-code strands should be released and identified on a microarray, this method is more direct. Moreover, due to the selective immune reaction and the dual bar-code mechanism, the resulting method could detect the two targets simultaneously. Multiple analysis in human serum was also performed, suggesting that our strategy was reliable and had a great potential application in early clinical diagnosis.


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INTRODUCTION Cytokines are soluble, low molecular weight proteins secreted by immune cells. These proteins often act as signal molecules to mediate immune responses, leading to recruitment and activation of lymphocytes at sites of injury or infectious.1 Recently, signaling through cytokines has been suggested to impact tumor cell survival, invasion and metastasis.2,3 These cytokines have been regarded as biomarkers in cancer diagnosis and treatment.4 However, single tumor marker is not specific and sensitive enough to meet the strict diagnosis in clinic because a marker may represent a variety of cancers and most cancers have more than one marker.5,6 Thus, using multiple biomarkers to investigate one kind of cancer is more reliable in practice.6 The commonly used methods to detect secreted cytokines are enzyme-linked immonosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay.7-14 These assays rely on the capture of cytokines by the specific antibody and then detect by a second antibody in association with either an enzyme-based or a fluorophore-based signal output. However, due to the stoichimetric ratio of target and signal molecule is 1:1, the sensitivity of these methods is relatively low.10 Moreover, in the case of multiplexed analysis, ELISA and ELISPOT suffer from the false positive signals, overlapping spectral features, nonuniform photobleaching rates and complex apparatus for signal readout.10-14 To circumvent these limitations, amplification approaches such as multimolecule labeled nanoparticles,15-17 nanowaires,18,19 bar-code strategy21,22 and hybridization chain reaction6,23,24 have been introduced to improve the sensitivity and signal intensity. Among them, bar-code strategy exhibits powerful amplification ability and has been widely used in sensitive detection of biomarkers.25-28 Bar-code assay is a sensitive strategy that takes advantages of short oligonucleotides as the surrogate targets in biological detection. Classical bar-code assay contains two types of particles. One is magnetic and the other is metallic. They are all modified with recognition units, which can sandwich the target with each other.21,22,30 The latter particle also carries a large quantities of oligonucleotide strands,



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which are referred to as bar-code strands. Due to the enrichment of the particle, a small amount of targets can be converted to a large numbers of bar-codes and this assay exhibits highly sensitivity for protein and nucleic acid targets.21,22,30,31 Mirkin’s group has constructed a multiplexed detection method for protein cancer markers based on the bar-code-based amplification strategy with the detection limit of 170 pM.31 But because the concentrations of protein biomarkers in body fluids are extremely low, the sensitivity of this method still cannot meet the practical application. Moreover, the bar-code strands should be released and recognized on a microarray, which may causes cross hybridization and improve the complexity. Thus, in this work, we developed a new multiplexed detection method for cytokines based on dual bar-code strategy and single-molecule counting. Single-molecule counting is a new kind of quantitative method that achieved by counting the target one by one.32-34 In the proposed method, we choose interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) as model analytes, which were correlated with a variety of physiological and pathological processes, such as latent tuberculosis, HIV infection and melanoma1,35,36. Firstly, the magnetic nanoprobe was fabricated with the second antibody and primary bar-code strands. Through the specific reaction of the second antibody and the antigen that fixed by the primary antibody, sandwich-type immunocomplex was formed on the substrate. Next, the primary bar-code strands acted as amplification units triggered multibranched hybridization chain reaction (mHCR)37, producing nicked double-stranded polymers with multiple branched arms, which were served as secondary bar-code strands. Subsequently, we specially designed a multimolecule labeled fluorescence probe that contains three fluorescent molecules and an overhang single-stranded domain. The secondary bar-code strands could hybridize with the multimolecule labeled fluorescence probes and generate enhanced fluorescence signals. Finally, the numbers of fluorescence dots were counted one by one for quantification. In this method, by integrating the primary and secondary bar-code-based amplification strategy, a small amount of targets can converted to a large mumbers of secondary bar-code strands, leading to a remarkable amplification. Moreover, the multimolecule labeled fluorescence probes can induce


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prominent enhancement of the fluorescence signal and further improve the sensitivity. The detection limits of the two targets INF-γ and TNF-α were both 5 fM. Unlike the typical bar-code assay that the bar-code strands should be released and recognized on a microarray, this method is more direct. In addition, due to the selective immune reaction and the dual bar-code mechanism, the resulting method exhibited an excellent specificity that could detect the two targets simultaneously. Importantly, multiple biomarker analysis in human serum was also performed, suggesting that our strategy was reliable and had a great potential application in early clinical diagnosis.

EXPERIMENTAL SECTION Materials and Reagents Human tumor necrosis factor-α (TNF-α), recombinant human interferon-γ (IFN-γ), polyclonal mouse anti-human tumor necrosis factor-α (anti-TNF-α), human IFN-γ antibody (anti- IFN-γ), human monoclonal biotinylated rabbit anti-human tumor necrosis

factor-α

(Bio-anti-TNF-α),

human

IFN-γ

biotinylated

antibody

(Bio-anti-IFN-γ) and human immunoglobulin G (IgG) were purchased from Abcam (Shanghai, China). Streptavidin-Magnetic NanoBeads (Streptavidin-MNBs) (350 nm diameter, 0.05% Tween-20, and 10 μL EDTA at a concentration of 3.324×1011 beads mL-1, 1.343 g mL-1, aqueous suspension containing 0.1% bovine serum albumin ) were purchased from Bangs Laboratories Inc (Fishers, IN). Bovine serum albumin (BSA, >98%) and uric acid (UA) were acquired from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Dopamine (DA) was purchased from Aladdin (Shanghai, China). Carcinoembryonic antigen (CEA) was acquired from Linc-Bio Science Co. Ltd. (Shanghai, China). Microscope cover glasses (22×22 mm2) was obtained from Cole-Parmer (Illinois, USA). The ELISA kits were purchased from Quantikine (Minneapolis, USA). All other chemicals (analytical grade) were purchased from standard reagent suppliers. All solutions were prepared using ultrapure water that was obtained by a Millipore Milli-Q water purification system (>18.25 MΩ). Clinical serum samples were obtained from the Hospital of Shandong University, China. The oligonucleotides (shown in Table S1)



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were synthesized and purified by Invitrogen (Beijing, China). Apparatus EFM imaging was performed with an Olympus IX81 fluorescence microscope (Tokyo, Japan) equipped with a high-numerical-aperture 60 × (1.45 NA) oil-immersion objective lens, a mercury lamp source, a mirror unit consisting of a 470-490 nm excitation filter (BP 470-490), a 505 nm dichromatic mirror (DM 505), a 510-550 nm emission filter (IF 580), and a 16-bit thermoelectrically cooled electron multiplying charge coupled device (EMCCD) (Cascade 512 B, Tucson, AZ, USA). Imaging acquisition was performed with the MetaMorph software (Universal Imaging, Downingtown, PA, USA). All captured images were then further processed with the Analyze Particles function in the public-domain image-processing software (ImageJ) to determine the number of single fluorescence particle counting. Preparation of the Antibody Coated Glass Substrate The epoxy-functionalized glass substrates were prepared according to the previous protocol.24,34,41 The freshly prepared substrate was incubated with 50 μL antibodies (anti-IFN-γ and anti-TNF-α, 0.01 nM) overnight at 37 °C. Then, the antibody coated substrate was blocked with 5% (w/v) BSA for 6 h at 37 °C. After each step, the substrate was rinsed three times with PBS-T (0.15 M NaCl, 7.6 mM Na2HPO4, 2.4 mM NaH2PO4, 0.05% Tween-20, PH 7.4) and PBS (0.15 M NaCl, 7.6 mM Na2HPO4, 2.4 mM NaH2PO4, pH 7.4) to remove the redundant agents. Preparation of the Magnetic Nanoprobes First, 2 μL streptavidin-MNBs were washed with 200 μL TTL buffer (100 mM Tris, 1 M LiCl, 0.1% Tween-20, pH 8.0) five times with the aid of an external magnet and resuspended in 60 μL PBS. Then, 10 μL bio-strand (1-bio-strand or 2-bio-strand, 0.2 μM), 10 μL bio-Antibodies (bio-anti-IFN-γ or bio-anti-TNF-α, 0.1 nM) and 30 μL streptavidin-MNB were incubated for 5 h at 37 °C. After that, the suspension was washed three times with 200 μL PBS under the external magnet. The resulting product, termed magnetic nanoprobes, was finally resuspended in 50 μL for the succeeding experiments.


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Assembly and Electrophoresis Analysis of the Multimolecule Labeled Fluorescence Probe Three fluorescence labeled single-stranded DNA (1-Ca, 1-Cb, 1-Cc or 2-Ca, 2-Cb, 2-Cc) and a longer single-stranded DNA were diluted in TE buffer (10 mM Tris, 1 mM Na2EDTA, pH 8.0), yielding a final concentration of 10 μM. Then, 10 μL of each strand were mixed with 60 μL TM buffer (20 mM Tris, 50 mM MgCl2, pH 8.0). Subsequently, the mixture was heated to 95 °C for 2 min and cooled to 30 °C (annealing) or cooled in ice bath (quenching) immediately. The final concentration of the fluorescence probes was 0.1 μM. In order to verify the assembly of this fluorescence probe, a polyacrylamide gel electrophoresis (native-PAGE) experiment was performed. The fluorescence probes were run on 12% native-PAGE gel in TAE-Mg buffer at 15 °C with a current of 30 mA. The Construction of the Multiplexed Assay Based on the Dual Bar-code Strategy and Single Molecule Detection The antibodies coated glass substrates were incubated with 50 μL targets (IFN-γ and TNF-α) at 37 °C for 30 min in humidity. After washing three times with PBS-T and PBS, respectively, 50 μL magnetic nanoprobes suspension acquired above were added onto the substrate and incubated at 37 °C for 35 min. The uncombined were removed from the substrate in the presence of external magnet. Following that, the substrate was washed with 50 μL PBS-T and PBS, respectively. The magnetic separation was repeated five times. Then, the cleaned substrate was incubated with a mixture of 10 μL H1 (1-H1 and 2-H1, 0.2 μM), 10 μL H2 (1-H2 and 2-H2, 0.22 μM) and 30 μL HCR buffer (50 mM Na2HPO4, 0.5 NaCl, pH 6.8) for 3 h. These hairpins were all heated to 90 °C for 5 min and cooled to 30 °C prior to use. After washing thrice with PBS-T and PBS, respectively, 50 μL fluorescence probe was added to the detection cell and incubated for 3 h at 37 °C. Subsequently, the substrate was rinsed three times with PBS-T and PBS to remove the nonhybridized fluorescence probes. Before fluorescence imaging with epi-fluorescence microscopy, 50 μL PBS was added.



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Fluorescence Imaging Fluorescence imaging was performed with the Olympus IX81 fluorescence microscope, which equipped with a 16-bit thermoelectrically cooled EMCCD, a mercury lamp, a mirror unit consisting of a 633 nm excitation filter (BP 633) and a >580 nm emission filter (IF580) for IFN-γ and a mirror unit consisting of 470 to 490 nm excitation filter (BP 470-490) and a >580 nm emission filter (IF580) for TNF-α. The images corresponding to different locations were obtained by manually moving the XY sample stage and analyzed by MetaMorph software.

RESULT AND DISCUSSION The Rational of the Proposed Method As illustrated in Scheme 1, firstly, the antibodies of IFN-γ and TNF-α were immobilized on the silanizated glass substrate by the epoxy-hydroxyl reaction. Meanwhile, streptavidin-MNBs were functionalized with the second antibody and oligonucleotide strands, forming the magnetic nanoprobes. The antibodies play the role of molecule recognition and the oligonucleotide strands are termed as primary bar-code stands. Then, the targets and magnetic nanoprobes were successively added onto the glass substrate. Through the immunoreactions of the antibodies and the antigens, sandwich-type immunocomplexes were formed on the substrate. Next, the primary bar-code strands served as amplification units triggered mHCR, producing nicked double-stranded polymers with multiple branched arms. Subsequently, the multimolecule labeled fluorescence probes were designed with three fluorescence molecules and a hanging single-stranded domain. Finally, the branched arms (termed secondary bar-code strands) hybridized with the fluorescent probes, generating fluorescence signals. Using the epi-fluorescence microscope with different channel, the numbers of fluorescence dots were counted one by one for quantification. Different with the commonly used methods, the cytokines detection in this method was converted to bar-code strands detection through the dual bar-code amplification strategy and the abundant of surrogate targets were hybridized with the fluorescence probes. Eventually, the stoichimetric ratio of target and signal was 1:n, which was


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helpful to improve the signal intensity and sensitivity.

Scheme 1. Schematic illustration of multiplexed detection of cytokines based on dual bar-code strategy and single-molecule counting. Design of the Initiators, Hairpins and Connectors In order to adapt mHCR to detect cytokines in a multiplexed manner, we designed two different oligonucleotides strands (1-bio-strand and 2-bio-strand; Table S1) and two pairs of hairpins (1-H1, 1-H2 and 2-H1, 2-H2; Table S1) based on the previous reports about HCR.23,37,38 Each pair of complementary hairpins can hybridize selectively with their respective initiator. Moreover, we designed two sets of connectors (1-Ca, 1-Cb, 1-Cc, 1-Cd and 2-Ca, 2-Cb, 2-Cc, 2-Cd; Table S1) for the self-assembly of multimolecule labeled fluorescence probes. Each set of connectors was synthesized with nonoverlapping fluorescent dyes (Cy5 and FAM) to empower multiplexed analysis. Self-assembly and Characterization of the Multimolecule Labeled Fluorescence Probe In this work, we designed a new fluorescence probe labeled with three fluorescent molecules, called multimolecule labeled fluorescence probe. Specially, four singlestranded DNA were assembled into a cruciform structure, which has a hanging single stranded sequence at one of the vertex. Other three vertexes were labeled with fluorescent dyes to generate fluorescence signals. To demonstrate the self-assembly of the fluorescence probe, the cruciform probes



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were analyzed using polyacryamide gel electrophoresis. As shown in Fig 1, strip 5 and 10 were the probes acquired by quenching and annealing, which were moved more slowly than the single strand DNA and any other combinations lacking one or more strands. This confirmed the successful assembling of the probes.39, 40 In addition, it is obvious that the assembly yield of fluorescence probes acquired by annealing is much higher than that by quenching. Thus we selected the latter for the subsequence experiments.

Fig. 1 Electrophoretic analysis of the fluorescence probes, 1-5 line stand for the electrophoretic

image

of

sequences

(1-Cd,

1-Cd+1-Cc,

1-Cd+1-Cc+1-Cb,

1-Cd+1-Cc+1-Ca and 1-Cd+1-Cc+1-Cb+1-Ca) acquired by quenching, 6-10 line stand for the electrophoretic image of the same sequences acquired by annealing, M is the marker. Optimization of the Reaction Conditions The reaction conditions played important roles for the assay such as the reaction time and component concentrations. To achieve the best performance of the proposed method, we studied the experimental parameters using the numbers of bright dots as standard. The blocking time of BSA, the concentration of bio-strands, bio-antibody, H1 and H2, the reaction times of each step were optimized. Fig. S1 depicted typical fluorescence response in correlation to theses assay conditions. The blocking time of BSA has a crucial effect on the background produced by the magnetic probes at the absence of antigens. Thus we varied the blocking time of BSA form 1 h to 8 h. As shown in Fig. S1A, the number of bright dots generated by the magnetic probes at the


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absence of antigens had a sharp decrease before 5 h of incubation and achieved a constant value after 6 h. So we chose 6 h as the optimized blocking time for BSA. Moreover, we optimized the reaction times of each step. As shown in Fig. S1B-E, the numbers of bright dots reached the maximum when the reaction time of the targets, magnetic probes, mHCR and fluorescence probes were 30 min, 35 min, 3 h and 3 h, respectively. The influences of concentrations of bio-strands, bio-antibody, H1 and H2 were also investigated. The results shown in Fig. S1F-I indicated that the optimized concentrations of bio-strands, bio-antibody, H1 and H2 were 0.2 μM, 0.1 nM, 0.2 μM and 0.22 μM, respectively. Quantification of IFN-γ and TNF-α with the Proposed Method Under the optimal conditions, IFN-γ and TNF-α concentration were quantified by counting the numbers of bright dots corresponding to a single molecule on the fluorescence images. The numbers of fluorescent dots in 10 subframe images of all positive and negative experiments were counted. The typical fluorescence images IFN-γ and TNF-α at different concentration in the range from 160 fM to 5 fM are shown in Fig. 2. It was obvious that in the lower concentration, the fluorescence intensity of bright dots was uniform. But when the concentration exceeded 120 fM, the bright dots became irregular and the fluorescence intensity of the bright dots was 2~3 times higher than the mean intensity of bright dots with lower concentrations. The possible reason was that the distance of two neighbor targets was small at the higher concentration. The bright spots generated by two nearest neighbor targets aggregated one brighter spot due to the diffraction of the fluorescence image.41 Therefore, when the concentration of the targets was exceeded 120 fM, it was unsuitable to count the dots one by one for quantification.



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Fig. 2 Fluorescent images of IFN-γ (red) and TNF-α (green) molecules at different concentrations. As shown in Fig. 3, there was a good linear relationship between the dots numbers and the concentration of targets over the range from 120 fM to 5 fM. The linear regression equation for IFN-γ and TNF-α were Y1=2.829+7.578C1 (R=0.998) and Y2=2.102+7.253C2 (R=0.997). The detection limits of IFN-γ and TNF-α were both 5 fM. Compared with other reported assays, this proposed method had a comparable or superior sensitivity for cytokine detection.6,23,31 The improvement of sensitivity should attributed to the dual bar-code-based amplification strategy and the multimolecule labeled fluorescence probe. In this method, the high end of the linear range is limited, because the probability that two or more than two molecules aggregated on the substrate increased.32,41-43 By taking more than 10 subframe images or expanding the area of the subframe, and taking the sum of the numbers of the bright dots in these images as the signal, the low end of the linear range can be reduced.32


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Fig. 3 Linear relationship between the numbers of bright dots and the targets. The error bar represents the standard deviation of three repetitive measurements. Selectivity, Precision and Recovery of the Proposed Method The selectivity was investigated to evaluate the feasibility of the proposed method in biological samples. Five proteins including BSA, thrombin, Ig G, APF and CEA were used as the possible interferences to evaluate the selectivity of the proposed method. As displayed in Fig. 4, at the same concentration, the dot numbers of BSA, thrombin, Ig G, APF and CEA were much lower than that of IFN-γ and TNF-α. These results indicated that this method had good selectivity in multiplexed detection of cytokines and could recognize the targets accurately.



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Fig. 4 (A) Images of the proposed method for different targets. (B) Specificity of the proposed strategy to IFN-γ and TNF-α by comparing them to the possible interference at 50 fM, respectively. 1-BSA; 2-thrombin; 3-Ig G; 4-APF; 5-CEA; 6-IFN-γ and TNF-α. As important parameters to appraise the precision of the method, the repeatability and reproducibility of the proposed method were investigated by the relative standard deviation (RSD) of intra- and inter-assay (n=3), as shown in Table S2 and Table S3. It was suggested that this method was reliable with acceptable precision. In addition, the recoveries of IFN-γ and TNF-α by spiking the standards into human serum were assessed to investigate the reliability for clinical application of this method. In these experiments, the human serum that diluted 10 times with PBS was selected as the blank serum. As shown in Table. S4, the Cadded was the added concentration, and the Cdetected was the detected concentration that has subtracted the blank. The recoveries of three samples (10 fM, 50 fM and 80 fM) were in the range of


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93-107% and 95-99%, respectively. The corresponding fluorescence images were shown in Fig. S2. Moreover, the spike-in experiments in human serum have also been performed throughout the concentration ranges that have been reported in human serum (as indicated in Fig. S3 and Table S5). These results suggested that our method was responsible for cytokines detection in human serum. Matrix Interference Experiment of This Method To estimate the possibility and reliability for clinical application of this assay, the interference effect of sample matrix components was first examined. Several components that might exist in the normal human serum were selected as the interferences, such as Na+, Cl-, glucose (GLU), uric acid (UA) and dopamine (DA). These interference components usually coexist in the normal human serum and the concentration of each component was close to the highest level in the normal human serum. As illustrated in Fig. 5, the numbers of bright dots generated by IFN-γ and TNF-α with different interference components were comparable to that without any additive. It was indicated that the interferences effect of matrix components on the proposed method could be ignored.

Fig. 5 The interference effects of various matrix components with 5 fM IFN-γ and TNF-α, respectively. 1-nomal; 2-Na+ (146 mM); 3-Cl- (180 μM); 4-GLU (6.11 mM); 5-UA (420 μM); 6-DA (0.8 nM). Multiplexed Imaging Analysis Multiple imaging analysis of the proposed method at different concentrations (10 fM, 50 fM and 100 fM) has been performed in different matrices. Fig. S4 and Fig. S5



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depicted the multiple imaging in buffer solution and human serum, respectively. To prevent the interference of endogenous cytokines, the serum was diluted 100 times with PBS buffer. These results indicated that this method was reliable for multiplexed detection of cytokines in both buffer system and complex system. Real Sample Analysis To further examine the application potentiality of the proposed strategy for clinical analysis, we tested three serum specimens. The concentrations of IFN-γ and TNF-α were measured by the proposed method and ELISA kit, respectively. In the proposed method, the serum samples have been diluted 10 times with PBS buffer before use. As displayed in Fig. 6, the concentrations of IFN-γ and TNF-α in three samples detected by the proposed method were 1.04-1.07×10-13 mol/L and 3.29-5.26×10-13 mol/L, respectively, which were in accordance with the previous articles44,45. These results indicated that our strategy was reliable and had a great potential application for the multiplexed analysis of cytokines in clinical samples.

Fig. 6 (A) Images of human serum determinations through the proposed method. (B) Cytokines determination by the proposed method and ELISA kit.


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CONCLUSION In summary, we have demonstrated a highly sensitive multiplexed detection method for cytokines based on dual bar-code strategy and single-molecule counting. To our knowledge, it is the first time to employ the bar-code strategy into single-molecule quantification. In this method, firstly, the individual target can be unambiguously quantified under complex conditions, which is attributed to the selective immune reaction and the dual bar-code mechanism. Secondly, by introducing the dual bar-code-based amplification strategy and the multimolecule fluorescence probes, a high sensitivity has been achieved and the detection limits are both 5 fM. Unlike the typical bar-code assay that the bar-code strands should be released and detected on a microarray, the proposed method is more direct. Finally, multiple biomarker analysis in human serum was performed, suggesting that our strategy was reliable and possessed the capacity in determination of low abundant biomarkers for clinical diagnosis.

ASSOCIATED CONTENT Supporting Information DNA sequences (Table S1), the optimization of reaction conditions (Fig. S1), the investigation of repeatability and reproducibility (Table S2 and Table S3), the spike-in experiments (Fig. S2, Table S4 and Fig. S3, Table S5), the multiple imaging analysis (Fig. S4 and Fig. S5). This material is available free of charge via the Internet at Http://pubs.acs.org.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, 21375078 and 21475077).



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