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Article Cite This: ACS Omega 2019, 4, 6210−6217
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A Highly Sensitive Capillary-Based Immunosensor by Combining with Peroxidase Nanocomplex-Mediated Signal Amplification for Detection of Procalcitonin in Human Serum Rongbin Nie,† Xuexue Xu,† Xiujun Cui,† Yiping Chen,*,‡ and Li Yang*,† †
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Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China ‡ College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China S Supporting Information *
ABSTRACT: While signal amplification is a promising approach for improving the detection sensitivity, it has never been applied in immunoassays with capillary-based sensors. In this study, we construct a sensitive short-capillary immunosensor for determination of procalcitonin (PCT) in human serum by introducing a robust signal amplification system based on a biotin−streptavidin-mediated peroxidase nanocomplex. We show that the proposed sensor combines essential advantages for immunoassays, including less sample consumption, high detection sensitivity, and high specificity. The proposed immunosensor for detection of PCT provides a large linear response range from 2.5 to 8.0 × 104 pg/mL with a low limit of detection of 0.5 pg/mL. The method is successfully applied for PCT determination in human serum samples, and the results are satisfactory with the clinical method. By combining with a signal amplification system that enhances immunoassay sensitivity significantly, the proposed peroxidase nanocomplex-mediated capillary-based sensors should have great potential value in detection of PCT and other lowlevel biomarkers.
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INTRODUCTION Combining high specificity of immunological reaction with sensitivity and convenient operation, immunosensors or miniaturized immunoassay devices have emerged as important assays for quantitative detection of biomarkers in clinical diagnosis. 1−7 Among various kinds of immunosensors, capillary-based immunosensors, which employ fused silica microcapillary tubes as support for bioreagent immobilization, have been developed rapidly in recent years.4,8−16 Compared to other solid-material-based sensors, capillary-based immunosensors show several essential merits for immunoassays. It is well known that assays based on microcapillaries offer low sample/reagent consumption, fast mass-transfer rate, and short analysis time.8,11 In addition, the capillary wall possesses an excellent light-wave-guiding property; thus, capillary-based immunosensors are especially suitable for sensitive optical detection such as chemiluminescence (CL) or laser-induced fluorescence.8,11,14,17 Moreover, capillary-based immunosensors feature the advantage of easy integration and automatization, which meet the requirements for miniaturized bioanalysis nowadays. A crucial issue that hampers further application of these promising capillary-based immunosensors for clinical diagnosis is their limited detection sensitivity, which can be attributed to the inherent one-dimensional cylindrical geometry of capillary tubes. To overcome this issue, three-dimensional capillary© 2019 American Chemical Society
based immunosensors were developed by placing eight capture antibody-coated capillary tubes inside a quartz tube or by coating the antibody on a photonic crystal fiber, which can be viewed as analogous to a bundle of capillary tubes.13,18 Another efficient approach is modification of the capillary surface via surface chemistry, for example, a porous-structured layer, to increase the surface-area-to-volume ratio for antibody immobilization. Generally speaking, the basic idea of all these elaborated studies is to increase the capillary surface area, allowing more amount of the antibody immobilized, thus increasing the detection sensitivity of the sensors. With either sophisticated design and manipulation or complicated surface modification, the detection sensitivity of these sensors can be improved by 3- to 10-fold compared to that of a normal capillary-based sensor.13,14,18 A promising strategy to further enhance the detection sensitivity of microcapillary-based sensors is to combine them with an effective signal amplification system, which has been adopted to improve their analytical performance in various fields.19−27 Surprisingly, to the best of our knowledge, up to now, there is no report about the application of a signal amplification system in capillary-based immunoassays. Very Received: January 27, 2019 Accepted: March 12, 2019 Published: April 3, 2019 6210
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Figure 1. Characterization of the surface of the capillary. (A) UV−vis spectrum of bare capillary (black line), BSA (red line), and BSA-coated capillary (blue line). (B) FT-IR spectrum of bare capillary (black line), BSA (red line), and BSA-coated capillary (blue line). (C) AFM image of the bare silica capillary surface. (D) AFM image of the silica capillary surface after Ab1 immobilization.
of the capillary, we selected BSA as a test protein. We first characterized a BSA-modified capillary using both UV−vis spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Figure 1A,B shows the UV−vis and FT-IR spectra, respectively, for the free BSA solution, a BSA-modified capillary, and a bare capillary. It is obvious that after modifying the capillary with BSA, an absorption peak at 280 nm appears in the UV−vis spectrum, which is identical to that of free BSA. The FT-IR spectrum of either the bare capillary or the BSAcapillary exhibits two strong absorption bands centered at 1096 and 806 cm−1 corresponding to vibrational frequencies of Si− O−Si asymmetric stretching and Si−O−H stretching motions, respectively, and a broadband at 3476 cm−1 that can be assigned to the vibration of the hydroxyl bond. After BSA immobilization, additional peaks at 1649 and 1519 cm−1 appear in the spectrum. These two peaks are characteristic vibrational bands of proteins, that is, the CO stretching from the primary amide and the N−H bending from the secondary amide of a peptide. The results indicate the successful immobilization of proteins on the surface of the capillary via a GA cross-linking strategy. Comparing the atomic force microscopy (AFM) image of an Ab1-immobilized capillary to that of a bare capillary provides further proof for the successful immobilization of Ab1 on the surface of the capillary (Figure 1C,D). Optimization of Response of the BS-Capillary-Based Immunosensor. The effect of several essential factors involved in the immunoassay was investigated. We optimized the immune reaction time in the BS-capillary-based immunosensor. In general, such immune reaction time is controlled by the mass transport of immunoreagents and the kinetic characteristics of immunoreactions. As shown in Figure 2A, the CL intensity increases as the incubation time is increased
recently, we developed a peroxidase nanocomplex-amplified signal amplification system based on a one-step self-assembly approach of streptavidin and biotin−peroxidase and combined it with a microfluidic chip sensor, effectively showing amplifying chemiluminescent signals for ultrasensitive and quantitative immunoassays.27 Here, we will extend our study to improve the detection sensitivity of capillary-based immunosensors based on our peroxidase nanocomplex-amplified signal amplification system. The proposed biotin−streptavidinmediated (BS) capillary-based immunosensor (BS-capillarybased immunosensor) is applied for the sensitive and quantitative detection of procalcitonin (PCT), whose amount level is elevated in many conditions, leading to systemic inflammatory response syndrome (e.g., bacterial infection).28−31 The results show that with introducing the signal amplification to the capillary-based immunosensors, up to two orders of magnitude improvement in the detection sensitivity can be achieved, without any additional manipulation of the capillary surface. The proposed study provides a simple but robust approach that should pave the way to the application of capillary-based immunosensors for rapid and sensitive detection of biomarkers.
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RESULTS Characterization of BS-Capillary-Based Immunosensor. To immobilize the capture antibody (Ab1), the inner surface of the capillary was first modified with primary amine by introducing 3-ADMS into the capillary, followed by adding GA as the cross-linking agent. One of the aldehyde groups would be immobilized onto the inner surface of the capillary through aldehyde amine condensation while leaving the other aldehyde group to condense with the amine group of Ab1. To verify the successful immobilization of Ab1 on the inner surface 6211
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Figure 2. Optimization of assay parameters for the BS-capillary-based immunosensor. (A) Incubation time of Ab1 and Ag. (B) Reading time of the CL signals. The concentrations of Ab1, Biotin-Ab2, SA, and Biotin-HRP were 40, 10, 1.25, and 12.5 μg/mL, respectively. (C) Concentration of Ab1. The concentration of Ag was 5 ng/mL, and those of Biotin-Ab2, SA, and Biotin-HRP were 10, 1.25, and 12.5 μg/mL, respectively. The incubation time of Ab1 and Ag was 60 min. (D) Concentration of Biotin-Ab2. The concentration of Ag was 5 ng/mL, and those of Ab1, SA, and Biotin-HRP were 40, 10, and 12.5 μg/mL, respectively. The incubation time of Ab1 and Ag was 60 min. (E) Ratio of SA to Biotin-HRP. The concentration of Ag was 5 ng/mL, and those of Ab1 and Biotin-Ab2 were 40 and 10 μg/mL, respectively. The incubation time of Ab1 and Ag was 60 min. (F) Concentrations of SA and Biotin-HRP. The concentration of Ag was 5 ng/mL, and those of Ab1 and Biotin-Ab2 were 40 and 10 μg/mL, respectively. The ratio of SA to Biotin-HRP was 1:4. The incubation time of Ab1 and Ag was 60 min.
from 10 to 60 min, and it remains to be unchanged after 60 min. Therefore, we chose 60 min as the incubation time for the determination of PCT antigen. The signal reading time was also investigated and optimized. Here, the reading time is related to the reaction time of CL substrate (luminol + H2O2) catalyzed by HRP. In Figure 2B, we show the CL intensity as a function of the reading time for detection of PCT with different concentrations (CPCT). For these measurements, the concentrations of Ab1, Biotin-Ab2, SA, and Biotin-HRP were 40, 10, 1.25, and 12.5 μg/mL, respectively. After 60 min immune reaction, the CL substrate was siphoned into the capillary, and the CL reaction was catalyzed by HRP labeled on the surface of the BS-capillary-
based immunosensor. The change of CL output was synchronously recorded and provided a changing curve of CL output over time. As shown in Figure 2B, for either CPCT, the CL intensity gradually increases as the reading time is increased and reaches a plateau at ∼250 s, which is the reading time used for the following immunoassays. The concentrations of Ab1 and Biotin-Ab2 (CAb1 and CBiotin‑Ab2, respectively) involved in the sandwich immunoassay are also essential parameters that may affect the response of the proposed BS-capillary-based immunosensor. Figure 2C presents the CL intensity measured with different CAb1 in the range of 1.0−100 μg/mL. It is easy to understand that the detection sensitivity of the sensor would be restricted if the 6212
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amount of antibodies immobilized on the capillary is insufficient. On the other hand, steric hindrance would be striking if CAb1 is too high, which may negatively influence the ability to capture targets and conduct nonspecific adsorption. Under the present assay conditions, the maximum response of the sensor was obtained at CAb1 = 40 μg/mL, as shown in Figure 2C. A similar tendency was observed for the curve of response versus CBiotin‑Ab2 (Figure 2D), resulting in the optimal CBiotin‑Ab2 of 10 μg/mL. Several parameters involved in the signal amplification system were also investigated to optimize the response of the BS-capillary-based immunosensor. First, we investigated the effect of the molar ratio of SA to Biotin-HRP by fixing the concentration of Biotin-HRP at 10 μg/mL while changing the concentration of SA from 0.01 to 50 μg/mL. The strongest CL signal was obtained when the concentration of SA was 1 μg/ mL (see Figure 2E), that is, the optimal molar ratio of SA to Biotin-HRP was 1:4. According to this molar ratio, the optimal concentrations of SA were investigated in the range of 0.83− 2.5 μg/mL. As shown in Figure 2F, the maximum CL intensity was obtained with the SA concentration of 1.25 μg/mL. Performance of the BS-Capillary-Based Immunosensor. Under the optimized conditions, we investigate the sensitivity and linear range of the BS-capillary-based immunosensor for PCT detection and compare the results with those obtained using a normal capillary-based immunosensor without signal amplification. Apparent enhancement in the response is observed, as shown in Figure 3A. The limit of quantitation (LOQ; the limit concentration at S/N = 10) of PCT using the BS-capillary-based immunosensor is as low as 0.5 pg/mL, showing 100 times improvement in sensitivity over that of the normal capillary-based immunosensor. The extremely low LOQ is attributed to the introduction of an SA−biotin system, which allows a large amount of HRP to be immobilized and thus amplifies the CL signal. The sensitivity of the capillary-based immunosensor with a signal amplification system meets the requirement for clinical diagnosis. In Figure 3B, we present the semilogarithmic plots of the data. An excellent linear response is obtained for PCT detection over a wide concentration range of 2.5 to 8.0 × 104 pg/mL (Y = 3761.7X + 13,932 (R2 = 0.9890)) using the BS-capillary-based immunosensor, while the linear range of the sensor without signal amplification is in the range of 50 to 8.0 × 104 pg/mL (Y = 353.8X − 15.7 (R2 = 0.9954)). The analytical performances of the BS-capillary-based immunosensor for detection of PCT are compared with other immunoassays (see Table S1 in the Supporting Information). It can be seen that compared to other immunoassays, the proposed BS-capillary-based immunosensor exhibits a larger linear range with a lower LOD, which will be significant for low-concentration PCT detection. High specificity is an important feature of immunosensors because the antigen−antibody reaction is a recognition behavior between biomolecules. In order to verify the selectivity of the proposed immunosensor, a series of different kinds of possible interferents including CRP, IL-6, AFP, ascorbic acid, glucose, Glu, Gly, K+, and Ca2+ were studied. The solutions of PCT (0.1 ng/mL) with interferents including CRP (5 μg/mL), IL-6 (7 pg/mL), AFP (25 ng/mL), ascorbic acid (5 μg/mL), glucose (2 mg/mL), Glu (28 μg/mL), Gly (18 μg/mL), K+ (200 μg/mL), and Ca2+ (100 μg/mL) or without interferents were assayed using the proposed immunosensor. The results are shown in Figure 4. The suppression or enhancement in signal intensity for PCT
Figure 3. Sensitivity of the BS-capillary-based immunosensors for PCT detection. (A) CL intensity for series concentrations of PCT. (B) Calibration curves of the BS-capillary-based immunosensors toward PCT standards at optimized conditions; the range of PCT concentrations is from 2.5 to 8 × 104 pg/mL. Every concentration level for three replicate measurements is shown. All measurements are done at room temperature.
Figure 4. Selectivity of BS-capillary-based immunosensors for detection of PCT. CRP, IL-6, AFP, ascorbic acid, glucose, Glu, Gly, K+, and Ca2+ were used as the interfering agents to evaluate the selectivity. The error bars represent the standard deviation from three assays (n = 3).
detection is in the range of −7.0% to +7.6% for all the interferents. The results indicate that the selectivity of the BScapillary-based immunosensor is acceptable, implying the feasibility for PCT detection in complex samples. The stability of the BS-capillary-based immunosensor was also investigated: the intra-coefficient of variation (CV) and the inter-CV are 6213
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amplifying the signal readout via a combination of the biotin− streptavidin signal amplification system with the capillarybased immunosensor. Such BS-capillary-based immunosensor is more robust since it can be achieved with the self-assembly of streptavidin and biotin−peroxidase to construct a streptavidin−biotin−peroxidase nanocomplex. The proposed method exhibits a large detection range from several picograms per milliliter to tens of nanograms per milliliter with an extremely low LOQ of 0.5 pg/mL, which makes it promising in sensing trace biomarkers in complicated real samples. Compared to a normal capillary-based sensor, up to two orders of magnitude enhancement in the detection sensitivity is achieved (see Figure 3), which is far beyond what can be achieved by any surface modification methods. One can expect that the sensitivity will be further improved by introducing the biotin−streptavidin signal amplification system into other more advanced capillary-based sensors, such as those reported in the literature.8,13 While PCT detection is performed in the present study, the proposed BS-capillary-based immunosensor can be applied for detection of different biomarkers, since the antibody immobilization is achieved using well-known silica chemistry and the biotin−streptavidin signal amplification system is compatible with the immunoassays. We also expect that the introduction of the biotin−streptavidin signal amplification system proposed in the study could be utilized for multiplex detection of biomarkers, as we have recently reported,14 which would stimulate further studies on the immunoassays based on capillary sensors.
below 6% and 11%, respectively, which indicates the good repeatability of BS-capillary-based immunosensor (Table S2 in the Supporting Information). Real Sample Analysis Using the BS-Capillary-Based Immunosensor. The analytical reliability of the proposed BScapillary-based immunosensor was investigated by recovery measurements of the spiked samples of healthy human serum. The results are presented in Table 1, with each data being an Table 1. PCT Recoveries in Serum Samples Using the BSCapillary-Based Immunosensors spiked value (ng/mL)
measured value (ng/mL)
recovery (%)
RSD (n = 3) (%)
0 0.1 0.5 2.0 5.0 10.0
0.119 0.215 0.667 1.880 5.265 8.145
96.0 109.6 88.1 102.9 80.3
10.7 7.3 5.1 2.7 6.8 1.8
averaged value of three repeatable assays. The recoveries for the spiked samples are 80.3%−109.6% with an RSD (n = 3) of 1.8%−10.7%, indicating that the BS-capillary-based immunosensor has a high accuracy for PCT determination in human serum samples. To further evaluate the immunosensor for practical applications, 15 clinical human serum samples were assayed, and the results were compared with those measured through the Roche-ECL method in the clinical laboratory of Beijing Friendship Hospital (see Table S3 in the Supporting Information). The scatter plot of the correlation between the two methods is demonstrated in Figure 5. The regression equation is Y = 0.9748X + 0.5460 (Y, BS-capillary-based immunosensor; X, Roche-ECL) with a correlation coefficient of 0.98. These results suggest that the developed immunosensor is reliable and accurate for detection of clinical biomarkers.
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CONCLUSIONS
In conclusion, the highly sensitive and quantitative capillarybased immunosensor combined with the peroxidase nanocomplex-amplified signal amplification system has been developed. The biotin−streptavidin signal amplification strategy efficiently enhanced the sensitivity of the capillarybased immunosensor, making the capillary-based immunosensor perfectly satisfying the demand for the trace biomarker in clinical diagnosis. Our study shows that the capillary-based immunosensor combined with a suitable signal amplification system is a feasible and efficient analytical platform, which has great potential applications in in vitro diagnosis.
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DISCUSSION The detection sensitivity of a capillary-based immunosensor is of vital importance for its application in clinical diagnosis. Unlike any of the existing methods that modify the capillary surface to increase antibody loading capacity and thus to enhance the detection sensitivity, our approach starts from
Figure 5. Scatter plot of the correlation between the assay results using the BS-capillary-based immunosensor and the Roche-ECL method. (A) Correlation analysis between the results of the BS-capillary-based immunosensor and Roche-ECL tests for PCT detection in 15 serum samples (R2 = 0.98). (B) Bland−Altman analysis for the agreement between the BS-capillary-based immunosensor and Roche-ECL tests of 15 serum samples. Data shows a 95% confidence interval of the mean. 6214
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Scheme 1. Working Principle of BS-Capillary-Based Immunosensors for Detection of PCTa
a (A) Ab1 immobilization. (B) Capture PCT human antigen (Ag) by the immobilized antibody after blocking the residue sites by GSSG. (C) Biotin-Ab2 and SA-Biotin-HRP nanocomplex immobilization to form multilayer construction for signal amplification. (D) Chemiluminescent signal readout.
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EXPERIMENTAL SECTION Reagents and Materials. Fused silica capillaries for fabrication of immunosensors (200 μm i.d., 365 μm o.d.) were obtained from Yongnian Optical Fiber Factory (Hebei, China). PCT samples (1.6 mg/mL), antibodies (capture antibody of PCT (Ab1, 5 mg/mL) and biotinylated antibody of PCT (Biotin-Ab2, 5 mg/mL)), and reagents for signal amplification (streptavidin (SA, 5 mg/mL) and biotinylated horseradish peroxidase (Biotin-HRP)) were provided by Sino Biological Inc. (Beijing, China). Other chemicals, including 3(aminopropyl)diethoxymethylsilane (3-ADMS), glutaraldehyde (GA; 50% aqueous solution), oxidized glutathione (GSSG), ascorbic acid, glucose, glycine (Gly), glutamic acid (Glu), interleukin 6 (IL-6), and C-reactive protein (CRP), were from Sigma-Aldrich Chemical (St. Louis, MO). CL detection was performed with the SW2010 Western Blotting ECL chemiluminescent kit (Beijing Solarbio Science & Technology Co., China). The clinical serum samples were obtained from the Hospital of Beijing (China). All other reagents were of analytical grade and used without further purification. A 10 mM phosphate-buffered saline (PBS) solution was prepared with Na2HPO4 and NaH2PO4; its pH value was adjusted to 7.4 with 0.1 M NaOH. BS-Capillary-Based Immunosensors. The principle for nanocomplex SA-Biotin-HRP signal amplification has been
described in detail in our previous study with some modifications.27 In brief, the enhanced CL signal is induced due to the fact that each SA molecule possesses four sites to bind a large amount of Biotin-HRP complex. The process of the proposed BS-capillary-based immunoassays are presented in Scheme 1, which can be accomplished via the following steps: (i) immobilization of Ab1 to form a capillary-based sensor, (ii) immune reaction between Ab1 and antigen (Ag), (iii) introducing SA-Biotin-HRP signal amplification, and (iv) chemiluminescent signal readout to determine the response of the immunosensor. The detailed description of the process is provided in the following. A bare fused silica capillary was first rinsed with 0.1 M HCl to remove any contaminants attached to the inner wall and then hydrophilized by rinsing with 0.1 M NaOH for 1 h at room temperature. After pretreatment, we burned the polyimide coating of the capillary to leave a 2 cm long window and then wiped the window with alcohol to maintain good light transmission. The capillary was then rinsed with 1% (v/v) 3-ADMS solution in toluene for 1 h. After washing with toluene and drying with nitrogen, the capillary was flushed with 2.5% (v/v) GA solution in water for 1 h and incubated for another 1 h, forming an aldehyde-activated inner surface for the immobilization of the antibody. After washing with water, the capture antibody of PCT (Ab1, 40 μg/mL) was injected into the capillary using a vacuum pump. (Unless otherwise 6215
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mL), glucose (2 mg/mL), Glu (28 μg/mL), and Gly (18 μg/ mL), K+ (200 μg/mL), and Ca2+ (100 μg/mL). The control sample was 10 ng/mL of PCT solution without interferents. All samples were assayed by the prepared immunosensor. For the recovery experiment, the PCT standard solution was spiked in the human serum sample and diluted to final concentrations of 0.1, 1.0, 2.0, 5.0, and 10.0 ng/mL. The human serum sample without spiking of PCT was used as the control. Real Sample Analysis. PCT levels in 15 clinical human serum samples were detected with the proposed BS-capillarybased immunosensors. An averaged result of detection repeated three times was used as the final value for each sample. The samples were without any purification or dilution steps. PCT concentrations were determined according to the calibration curve.
specified, the reagents were all injected using a vacuum pump (1500 kPa for 1 s), and the injection volume was controlled at ∼10 μL.) The capillary was kept at 4 °C overnight with both ends sealed to avoid evaporation of solvent. This allows antibody immobilization via the covalent bond between the amino group of Ab1 and the aldehyde group on the capillary surface. Any unbound Ab1 was removed by washing the capillary with PBS solution, and the residue sites was blocked by GSSG (10 mg/mL in PBS) for 2 h to eliminate nonspecific adsorption. After washing with PBS, the capillary was cut into several 6 cm long sections, each with a 2 cm long window. The prepared capillary-based immunosensors were kept at 4 °C whenever not in use. Measuring the response of the capillary-based immunosensor is achieved through a multilayer-type assay mode using Biotin-Ab2/SA/HRP-biotin as traces and H2O2/luminol as chemiluminescent substrates. Immune reaction was triggered by injecting PCT Ag standards or clinical samples into the capillary-based immunosensors with incubation for 1 h at 37 °C. The capillary was then washed with PBST (0.01 M PBS containing 0.05% (v/v) Tween-20, pH = 7.4) to remove any unspecific physically adsorption. To enhance the detection sensitivity, signal amplification was introduced by simply injecting Biotin-Ab2, SA, and Biotin-HRP into the sensor to form a three-layer structure with the aid of strong SA−biotin conjugation. The detailed process is as follows. First, 10 μg/ mL Biotin-Ab2 was injected and incubated for 1 h at 37 °C to construct a sandwich structure. Then, the solution of SABiotin-HRP nanocomplex containing 1.25 μg/mL SA and 12.5 μg/mL Biotin-HRP was introduced and incubated for 30 min at 37 °C, forming a multilayer immunocomplex on the inner surface of the capillary. Finally, the excess reaction solution was removed by rinsing with PBST. After washing three times with PBST, the liquid in the capillary was evacuated using a vacuum pump. To record the chemiluminescent signal, the immunosensor was inserted into the homemade capillary holder, and one end of the short capillary was immersed into the freshly prepared solution containing the chemiluminescent substrates. The immunosensor was filled with a luminol-based substrate via siphon effect and was immediately placed in a special stand in front of the photon counting detector in the light-tight box. Luminescence is generated by the reaction of luminol/H2O2, which is catalyzed by HRP in the inner surface of the capillary. The realtime chemiluminescent output in the immunosensor was recorded by a CH326 photon counting detector module (Beijing Hamamatsu Photon Technique Inc., Beijing). Data acquisition was performed using a CH297-01 counting unit and its software package (Beijing Hamamatsu Photon Technique Inc., Beijing). CL readings were integrated for 1 s, measuring a mean value of photon counts for 15 s. Results are presented as the mean and standard deviation of relative light units (RLU). The CL intensity is defined by subtracting the CL intensity of blank solution from the CL intensity of targets. For comparison, a normal capillary-based immunosensor without signal amplification was carried out to PCT detection using chemiluminescent readout produced under the HRPlabeled-Ab2 catalyzed reaction. Specificity and Recovery. To perform the specificity experiment, we prepared a series of PCT solutions (0.1 ng/ mL) containing different interferents including CRP (5 μg/ mL), IL-6 (7 pg/mL), AFP (25 ng/mL), ascorbic acid (5 μg/
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00249.
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Comparison of analytical performances, performance variations, and real sample analytical results (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (Y.C.). *E-mail:
[email protected] (L.Y.). Tel: +86-43185099762. Fax: +86-431-85099762. ORCID
Yiping Chen: 0000-0001-9309-2730 Li Yang: 0000-0001-6723-7377 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (grant nos. 21775017, 21475019, 81671784) and the Natural Science Foundation of Jilin Province, China (grant no. 20180101174JC). L.Y. would also like to thank the support from the Jilin Provincial Department of Education and Jilin Provincial Key Laboratory of MicroNano Functional Materials (Northeast Normal University).
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REFERENCES
(1) Zhou, Q.; Xue, H.; Zhang, Y.; Lv, Y.; Li, H.; Liu, S.; Shen, Y.; Zhang, Y. Metal-free all-carbon nanohybrid for ultrasensitive photoelectrochemical immunosensing of alpha-fetoprotein. ACS Sens. 2018, 3, 1385−1391. (2) Algaar, F.; Eltzov, E.; Vdovenko, M. M.; Sakharov, I. Y.; Fajs, L.; Weidmann, M.; Mirazimi, A.; Marks, R. S. Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients. Anal. Chem. 2015, 87, 8394−8398. (3) Fan, F.; Shen, H.; Zhang, G.; Jiang, X.; Kang, X. Chemiluminescence immunoassay based on microfluidic chips for α-fetoprotein. Clin. Chim. Acta 2014, 431, 113−117. (4) Moser, A. C.; Willicott, C. W.; Hage, D. S. Clinical applications of capillary electrophoresis based immunoassays. Electrophoresis 2014, 35, 937−955. (5) Konry, T.; Novoa, A.; Shemer-Avni, Y.; Hanuka, N.; Cosnier, S.; Lepellec, A.; Marks, R. S. Optical fiber immunosensor based on a
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poly(pyrrole−benzophenone) film for the detection of antibodies to viral antigen. Anal. Chem. 2005, 77, 1771−1779. (6) Liebes, Y.; Marks, R. S.; Banai, M. Chemiluminescent optical fiber immunosensor detection of Brucella cells presenting smooth-A antigen. Sens. Actuators B: Chem. 2009, 140, 568−576. (7) Jiang, C.; Alam, M. T.; Silva, S. M.; Taufik, S.; Fan, S.; Gooding, J. J. Unique sensing interface that allows the development of an electrochemical immunosensor for the detection of tumor necrosis factor α in whole blood. ACS Sens. 2016, 1, 1432−1438. (8) Momeni, N.; Ramanathan, K.; Larsson, P. O.; Danielsson, B.; Bengmark, S.; Khayyami, M. CCD-camera based capillary chemiluminescent detection of retinol binding protein. Anal. Chim. Acta 1999, 387, 21−27. (9) Torabi, F.; Mobini Far, H. R.; Danielsson, B.; Khayyami, M. Development of a plasma panel test for detection of human myocardial proteins by capillary immunoassay. Biosens. Bioelectron. 2007, 22, 1218−1223. (10) Surugiu, I.; Svitel, J.; Ye, L.; Haupt, K.; Danielsson, B. Development of a flow injection capillary chemiluminescent elisa using an imprinted polymer instead of the antibody. Anal. Chem. 2001, 73, 4388−4392. (11) Yang, Z.; Zong, C.; Ju, H.; Yan, F. Streptavidin-functionalized capillary immune microreactor for highly efficient chemiluminescent immunoassay. Anal. Chim. Acta 2011, 706, 143−148. (12) Cao, Y.-C. A capillary based chemiluminscent multi-target immunoassay. J. Fluoresc. 2015, 25, 563−568. (13) Yu, Q.; Wang, X.; Duan, Y. Capillary-based three-dimensional immunosensor assembly for high-performance detection of carcinoembryonic antigen using laser-induced fluorescence spectrometry. Anal. Chem. 2014, 86, 1518−1524. (14) Song, X.; Nie, R.; Liu, X.; Chen, Y.; Yang, L. Multiplex immunoassays using surface modification-mediated porous layer open tubular capillary. Anal. Chim. Acta 2018, 1043, 1−10. (15) Zhang, J.; Wang, S.; Liu, K.; Wei, Y.; Wang, X.; Duan, Y. Novel signal-enhancing immunoassay for ultrasensitive biomarker detection based on laser-induced fluorescence. Anal. Chem. 2015, 87, 2959− 2965. (16) Yu, Q.; Zhan, X.; Liu, K.; Lv, H.; Duan, Y. Plasma-enhanced antibody immobilization for the development of a capillary-based carcinoembryonic antigen immunosensor using laser-induced fluorescence spectroscopy. Anal. Chem. 2013, 85, 4578−4585. (17) Xu, M.; Qian, X.; Zhao, K.; Deng, A.; Li, J. Flow injection chemiluminescent competitive immunoassay for the β-adrenergic agonist salbutamol using carboxylic resin beads and enzymatic amplification. Sens. Actuators B: Chem. 2015, 215, 323−329. (18) Liu, X.; Song, X.; Dong, Z.; Meng, X.; Chen, Y.; Yang, L. Photonic crystal fiber-based immunosensor for high-performance detection of alpha fetoprotein. Biosens. Bioelectron. 2017, 91, 431− 435. (19) Qian, S.; Lin, M.; Ji, W.; Yuan, H.; Zhang, Y.; Jing, Z.; Zhao, J.; Masson, J.-F.; Peng, W. Boronic acid functionalized Au nanoparticles for selective microrna signal amplification in fiber-optic surface plasmon resonance sensing system. ACS Sens. 2018, 3, 929−935. (20) Lambert, A.; Yang, Z.; Cheng, W.; Lu, Z.; Liu, Y.; Cheng, Q. Ultrasensitive detection of bacterial protein toxins on patterned microarray via surface plasmon resonance imaging with signal amplification by conjugate nanoparticle clusters. ACS Sens. 2018, 3, 1639−1646. (21) Zhu, Q.; Chai, Y.; Zhuo, Y.; Yuan, R. Ultrasensitive simultaneous detection of four biomarkers based on hybridization chain reaction and biotin−streptavidin signal amplification strategy. Biosens. Bioelectron. 2015, 68, 42−48. (22) Fang, Y.-S.; Wang, H.-Y.; Wang, L.-S.; Wang, J.-F. Electrochemical immunoassay for procalcitonin antigen detection based on signal amplification strategy of multiple nanocomposites. Biosens. Bioelectron. 2014, 51, 310−316. (23) Li, N.; Chen, J.; Luo, M.; Chen, C.; Ji, X.; He, Z. Highly sensitive chemiluminescence biosensor for protein detection based on
the functionalized magnetic microparticles and the hybridization chain reaction. Biosens. Bioelectron. 2017, 87, 325−331. (24) Qin, Y.; Zhang, L.; Li, S.; Zhao, J.; Huang, Y.; Zhao, S.; Liu, Y.M. A microchip electrophoresis-based fluorescence signal amplification strategy for highly sensitive detection of biomolecules. Chem. Commun. 2017, 53, 455−458. (25) Choi, J.-W.; Jo, B.-G.; deMello, A. J.; Choo, J.; Kim, H. Y. Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA−protein interactions. Analyst 2016, 141, 6499− 6502. (26) Jiang, L.; Li, F.; Feng, J.; Wang, P.; Liu, Q.; Li, Y.; Dong, Y.; Wei, Q. An optionality further amplification of an sandwich-type electrochemical immunosensor based on biotin−streptavidin−biotin strategy for detection of alpha fetoprotein. RSC Adv. 2016, 6, 24373− 24380. (27) Wu, J.; Chen, Y.; Yang, M.; Wang, Y.; Zhang, C.; Yang, M.; Sun, J.; Xie, M.; Jiang, X. Streptavidin-biotin-peroxidase nanocomplexamplified microfluidics immunoassays for simultaneous detection of inflammatory biomarkers. Anal. Chim. Acta 2017, 982, 138−147. (28) Assicot, M.; Bohuon, C.; Gendrel, D.; Raymond, J.; Carsin, H.; Guilbaud, J. High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 1993, 341, 515−518. (29) Weglöhner, W.; Struck, J.; Fischer-Schulz, C.; Morgenthaler, N. G.; Otto, A.; Bohuon, C.; Bergmann, A. Isolation and characterization of serum procalcitonin from patients with sepsis. Peptides 2001, 22, 2099−2103. (30) Demissei, B. G.; Cleland, J. G.; O’Connor, C. M.; Metra, M.; Ponikowski, P.; Teerlink, J. R.; Davison, B.; Givertz, M. M.; Bloomfield, D. M.; Dittrich, H.; van Veldhuisen, D. J.; Hillege, H. L.; Voors, A. A.; Cotter, G. Procalcitonin-based indication of bacterial infection identifies high risk acute heart failure patients. Int. J. Cardiol. 2016, 204, 164−171. (31) Park, B. S.; Kim, S. E.; Park, S. H.; Kim, J.; Shin, K. J.; Ha, S. Y.; Park, J.; Kim, S. E.; Lee, B. I.; Park, K. M. Procalcitonin as a potential predicting factor for prognosis in bacterial meningitis. J. Clin. Neurosci. 2017, 36, 129−133.
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DOI: 10.1021/acsomega.9b00249 ACS Omega 2019, 4, 6210−6217
