Robust and multiplexed colorimetric immunoassay for cardiovascular disease biomarkers detection in serum with high specificity

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Robust and multiplexed colorimetric immunoassay for cardiovascular disease biomarkers detection in serum with high specificity Hai-dao Dong , Li-hong Huang , Dan-yang Liu , Lin Zhou , Zhen-hua Wu , Zu-le Cheng , Hui-ying Liu , Hong-ju Mao PII: DOI: Reference:

S0026-265X(19)31887-9 https://doi.org/10.1016/j.microc.2019.104334 MICROC 104334

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

23 July 2019 8 October 2019 11 October 2019

Please cite this article as: Hai-dao Dong , Li-hong Huang , Dan-yang Liu , Lin Zhou , Zhen-hua Wu , Zu-le Cheng , Hui-ying Liu , Hong-ju Mao , Robust and multiplexed colorimetric immunoassay for cardiovascular disease biomarkers detection in serum with high specificity, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104334

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Highlights 

A robust, simple, reliable and multiple-targeted colorimetric immunoassay platform.



Detection of CVD protein biomarkers in serum with high specificity.



Dual signal amplification based on gold deposition for colorimetric immunoassay.

Robust and Multiplexed Colorimetric Immunoassay for Cardiovascular Disease Biomarkers Detection in Serum with High Specificity Hai-dao Donga†, Li-hong Huangc†, Dan-yang Liua†, Lin Zhoub , Zhen-hua Wub, Zu-le Chengb,d, Hui-ying Liua*, Hong-ju Maob* a

School of Stomatology, Dalian Medical University, Dalian 116044, China

b

State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China c

The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, China

d

University of Chinese Academy of Sciences, Beijing 100039, China

†

Hai-dao Dong , Li-hong Huang and Dan-yang Liu contributed to this work equally.

*Corresponding Authors: Hui-ying Liu a*, E-mail: [email protected] School of Stomatology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, China. Tel: +86-41186110404; Fax: +86-41186110397; Hongju Mao b*, E-mail: [email protected] Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Science, No.865 Changning Rd. Shanghai, 200050, P.R. China

Tel: +86-02162511070; Fax: +86-02162511070-8714; Abstract Multiplexed detection of increased CRP and IL-6 levels in serum could warn the risk of Cardiovascular Disease (CVD) in clinical diagnosis. Herein, we report a robust, simple, reliable and multiple-targeted colorimetric immunoassay platform for the detection of CVD protein biomarkers in serum with high specificity. With dual signal amplification involving in AuNPs probes via ssDNA hybridization and AuNPs initiated gold deposition, this colorimetric immunoassay achieves a limit of detection (LOD) of 326 pg/mL for CRP and a LOD of 8 pg/mL for IL-6 simultaneously. In addition, this assay exhibited high specificity and selectivity when challenged with different interfering proteins. Clinical feasibility is evaluated by utilizing this assay in clinical serum samples of CVD patients and the experimental results show a good consistency with ELSIA kit. Importantly, this immunoassay shows several advantages such as simple operation, less time-consuming, multiplex, reliability and no requirement of labelled reagents and expensive equipment, which demonstrates its potential utility in clinical diagnostics for CVD.

Keywords: colorimetric immunoassay; multiple-targeted; CVD protein biomarkers; dual signal amplification; Gold deposition

1. Introduction

According to the statistical data reported by the World Health Organization (WHO), cardiovascular disease (CVD) has been the premier cause of death around the world, accounting for almost 18 million deaths and 31% of all global deaths annually.[1] Obviously, huge health and economic burdens has caused by CVD around the world. The presence of CVD risk factors always existed in the disease process of CVD that experiences over years from a subclinical state to clinical symptoms [2]. Consequently, the assessment of several risk factors such as unhealthy habits, circumstances, family history, blood cholesterol and baseline levels of blood pressure, was traditionally involved in the prediction of an individual’s CVD risk [3]. However, these risk factors alone in a model as prognostic tools are deficient in CVD risk prediction [4-6]. Biomarkers are better tool to screen high-risk individuals, to diagnose disease progression robustly and precisely, and to predict and treat patients with disease availably [7]. Cardiovascular biomarkers have the potential to aid in screening, diagnosis and assessment of prognosis of CVD disease. Elevated serum level of numerous individual biomarkers, such as C-reactive protein (CRP)[8], interleukin-6 (IL-6)[9], B-type natriuretic peptide (BNP)[8], etc., have been prove to associate with cardiovascular risk in ambulatory persons.

There was evidence that the enhancive availability of multiple biomarkers from different biologic pathways could predict the risk of cardiovascular events, and enhance risk stratification of ambulatory persons [10]. Especially, clinical studies have reported an association of increased CRP and IL-6 levels with higher risk of CVD and all-cause mortality [11-13]. What’s more, aggressive periodontitis would

also result in a significant increase in the levels of plasma CRP and IL-6[14]. Therefore, multiplexed detection of CRP and IL-6 levels in serum could warn the risk of CVD in clinical diagnosis, and bridge the gap between CVD and periodontitis for further disease-related study at the same time.

Several successful strategies have been utilized to achieve multiple detection of CRP and IL-6 levels. Enzyme-linked immunosorbent assay (ELISA) is recognized as the standard method for the quantification of biomarkers in serum, which has been widely used in the multiple detection of CRP and IL-6[13, 15, 16]. Beads colorimetric assay based multianalyte detection system was reported for the simultaneous detection of the CVD biomarkers, C-reactive protein and interleukin-6, in human serum samples [17]. Recently, a simple aqueous based microcontact printing method to pattern multiplexed bioassays for IL-6 and CRP with the limit of detection at nM level [18]. However, these methods involve sophisticated experimental techniques, time consuming labeling procedures, expensive reagents and experimental tools. In addition, interdigitated capacitor arrays were designed for label-free detection of C-reactive protein (CRP) and IL-6 with the limit of detection at pg/mL level [19]. But the sensitivity and specificity of this approach were not evaluated in the serum and it also need professional equipment. Therefore, a simple, low-cost and robust diagnostic tool for multiplex analysis of CVD biomarkers are urgently needed.

Protein microarrays are already established as practical and valuable multiplexed tools for diagnostic applications due to the advantage of miniaturization, high throughput, and robustness over conventional bioanalysis systems [20-26]. A novel protein

microarray for the early stage diagnosis of sepsis that allows the simultaneous detection of CRP, procalcitonin, and IL-6 has been developed [20]. In this study, on-chip calibration was used to increase the sensitivity, which weakened the utility of protein microarrays. The high accuracy of immunoassays method at low concentration especially in situation with low abundance proteins remains a challenge. Therefore, a simple, sensitive, and cost-effective amplification strategies may be a good candidate to enhance the performance of protein microarrays. Many elegant approaches, such as rolling-circle amplification [27], tyramide deposition based ELISA signal amplification [28], PEG based chemiluminescence microarray [29], Copper-free TCO-Tz click chemistry [30] and ZnO nanomulberry signal enhancement strategy [31], have improved the sensitivity of protein microarrays significantly. With the advantages such as robust and easy preparation, efficient surface modification, and desirable bio-compatibility, gold nanoparticles (AuNPs) have been utilized as labels for signal amplification and biomedical application [32-34]. To accomplish high sensitivity for the detection of biomolecules, amount of sensing amplification approaches have been designed through the use of AuNPs. Bio-barcode techniques [35], nanoparticle aggregates [36], gold-labeled silver stain method [37], and cuprous oxide nanoparticles [38], were successfully coupled with AuNPs to enhance the performance of colorimetric sensing. But the only usage in centrifugal tubes for some amplification strategies, complex procedure, biological toxicity and low signal noise

ratio, limit the clinical use of these current amplification methods in protein microarray.

Here, we propose a simple, high specific and multiple immunoassay chip with high signal to noise ratio for simultaneous detection of CRP and IL-6 in clinical serum. To amplify the signal of protein microarrays firstly, bifunctional AuNPs probes with antibodies and single stranded DNA (ssDNA) could accurately bind to the target molecules and then it hybridized with complementary ssDNA conjugated AuNPs. On the basis of this, AuNPs initiated gold reduction and subsequent deposition were utilized for second signal amplification. With this dual signal amplification, the excellent specificity and highly sensitivity for CRP and IL-6 simultaneous detection was achieved by using the proposed multiplexed immunoassay. Moreover, in order to verify the clinical applicability, this multiplexed immunoassay was evaluated in 28 clinical serum samples. Experimental data were in good agreement with those obtained by ELISA kit. Given the features of superior specificity, robustness and multiplex, this proposed method provides a promising tool for diseased-related low abundance proteins detection and the warning of the risk of CVD in clinical diagnosis.

2. Material and methods

2.1. Reagents and instruments

Anti-CRP capture antibody, anti-CRP detection antibody and CRP were purchased from Hytest Ltd. (Turku, Finland). Anti-IL-6 capture antibody, anti-IL-6 detection antibody and IL-6 were obtained from eBioscience Inc. (San Diego, USA). Gold

nanoparticles (AuNPs) of 15nm were synthesized by Shanghai Water Biotechnology (Shanghai, China). Tetrachloroauric(III) acid trihydrate was purchased from Acros organics (Geel, Belgium). Goat anti-mouse IgG, 4-morpholine ethane sulfonic acid (MES), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA) and Tween-20 from Sigma-Aldrich (Shanghai, China). Non-fat powdered milk was supplied by from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Microarray stabilizing solution was purchased from SurModics Inc. (Eden Prairie, USA). Aldehyde chips and spotting liquid were obtained from Shanghai Baiao Science and Technology Co., Ltd. (Shanghai, China). Clinical serum samples were obtained from the second hospital of Dalian medical university (Dalian, China). Samples were obtained with informed consent from patients. ELISA kits were purchased from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China). Other chemical reagents were purchased from Shanghai Ling-Feng Chemical Reagents Ltd. (Shanghai, China). Resuspension buffer (10 mM PBS, 0.2% Tween-20, 0.1% PVP, 1% BSA, and 2% sucrose, pH = 7.4) and gold deposition buffer (100 mM Tetrachloroauric(III) acid trihydrate; 30% H2O2; 25 mM MES (v:v:v, 5:3:2)) were prepared. Ultrapure water (18.2 MΩ.cm) was obtained from Milli-Q system (Millipore, Bedford, MA, USA). Two complementary single stranded DNA (ssDNA) were synthesized by TaKaRa Company (Dalian, China). The sequences of ssDNA1 and ssDNA2 are 5'-SH-(CH2)3-T10GCACAGGAGCAACAG-3' and 5'-SH-(CH2)3-T10CTGTTGCTCCTGTGC3', respectively.

Following instruments were used for different analyses: Prosys5510A type biochips spotter (Cartesion Technology Company, USA), FYY-3 type hybridization instrument (Xinghua Instrument Factory, China), BX51TRF microscope (Olympus Corporation, Japan), 5804R low temperature centrifuge and Thermostatic mixing device (Eppendorf AG, Germany), JA5003N electronic balance (Shanghai Precision Scientific Instrument Co. Ltd. China), V670 UV-visible spectrometer (Jasco Japanese Company, Japan), and JEM 2100 Transmission Electron Microscope (JEOL. Ltd., Japan)

2.2. Antibody microarrays fabrication

In the first step, CRP capture antibody, IL-6 capture antibody and goat anti-mouse IgG (quality control point) were mixed with spotting liquid separately (v/v 1:1) and then transferred to a plate. In the second step, the printing of antibody microarray on an aldehyde chip was performed with Prosys5510 type biochips spotter. The diameter of the microarray spots was about 80 m, and the interval between spots was 400 m. The third step involved placing the microarrays at room temperature (RT) for 24 h. In the fourth step, 40 L microarray stabilizing solution was added to block for 30 min at RT. The microarray stabilizing solution was then washed away, and the antibody microarray was dried at RT. Finally, the antibody microarrays were stored at 4 °C under desiccant and vacuum until use.

2.3. Preparation of the detection probes

The detection probes were prepared according to these following methods. Firstly, the pH of AuNPs solution (1 mL) was adjusted to 8.5 using 0.2 M K2CO3. For the preparation of antibodies and ssDNA functionalized AuNPs (ssDNA–AuNPs– antibodies), 1.1 L anti-CRP detection antibody (8.7 mg/mL) and 20 L Anti-IL-6 detection antibody (500 g/mL) were separately added into the 1 mL AuNPs solution and then placed at room temperature for 10 min. Subsequently 3 L of 100 M ssDNA1 was added and mixed. After stirring at RT for 20 h, 0.5 L tween-20 was added, and the mixture was incubated for 1 h. Next, the solution was subjected to “ageing” with adding 2 M NaCl; the final concentration of NaCl was 150 mM. After stirring the solution for another 20 h at RT, 10 L of 10% BSA was added and blocked for 15 min. The excess reagents were removed by centrifugation at 10,000 rpm for 50 min at 4 °C. The supernatant was discarded, and functioned AuNPs for detection were resuspended in 100 L resuspension buffer and stored at 4 °C until use.

2.4. Preparation of the signal amplification probes

The complementary ssDNA functioned AuNPs were used as the signal amplification probes. Firstly, 3.5 L of 100 M ssDNA2 was added into 1 mL AuNPs solution and incubated for 20 h at RT. Next, 0.5 L Tween-20 was added and maintained for 1 h. For “ageing”, 2 M NaCl was added into the mixture successively; the final concentration of NaCl was 150 mM. The mixture was then incubated for 20 h at RT. Finally, 15 L of 10% BSA was added and blocked for 15min. Unconjugated ssDNAs were removed by centrifugation at 10,000 rpm for 50 min at 4 °C, and the obtained

signal amplification probes were redispersed in 100 L resuspension buffer and kept at 4 °C until use.

2.5. The preparation of Colorimetric Immunoassay In the first step, 40 L of 1.5% non-fat milk solution was added to block remaining sites on the antibody microarray and incubated for 5 min. Next, 15 L antigen, 1 L CRP detection probe, 3 L IL-6 detection probe and 3 L signal amplification probe were mixed and added to the antibody microarray; then incubated for 40 min in hybridization instrument at 37 °C. Thereafter, the microarray was washed with deionized water three times to remove residual probes, followed by air drying. After air drying, 25 L gold deposition buffer was added to the microarray and incubated 8 min, followed by rinsing the microarray with deionized water three times to terminate the reaction; the entire process was performed inside the dark chamber. Finally, the images of the microarray were taken using a microscope and the data were analyzed using a Gray analysis 5.0 software (developed in our custom made software). Figure 1 illustrates the detection principle schematically.

Figure1. Schematic representation of the detection process of CRP and IL-6.

3.Results and discussion

3.1. Characterization of the probes

We characterized and monitored the detection probes and signal amplification probes using by UV-Vis spectrometer and transmission electron microscope (TEM, operate voltage: 120 V). Combining antibodies and ssDNA with AuNPs led to the change in AuNPs size, and this would result in a red shift of the maximum absorption peak of AuNPs in the absorption spectrum [39]. A remarkable absorption peak of AuNP was observed at the wavelength of 518.5 nm. And the peak shifts to 523 nm and 525.5 nm, after AuNP binding to IL-6 and CRP ssDNA detection probes, respectively. When the signal amplification probes ssDNA are assembled, the absorption peak moves to 523.5 nm. The UV-vis spectra presented in Fig. 2a clearly shows red shifts range from 4 nm to 7 nm, respectively, in the absorption peaks of the detection probes and the signal amplification probe. Figures 2b,2c show the TEM images of AuNPs, detection probe and signal amplification probe, respectively. We found the diameter of AuNPs as 15 nm with a good dispersion (Fig. 2b). The detection probe (obtained by combining antibody and ssDNA with AuNPs) and the signal amplification probe (obtained by combining ssDNA with AuNPs) did not change the stability of AuNPs, showing well-dispersed AuNPs for the two AuNPs probes (Figures 2c, 2d). In addition, the surfaces of the AuNPs probes presented with a halo compared with the

bare AuNPs. Hence, both UV-vis and TEM results confirm the successful labeling of ssDNA and antibodies onto the AuNPs surface.

Figure 2. (a) UV–vis absorption spectra, (b) TEM of AuNPs, (c) TEM of the detection probes, (d) TEM of the signal amplification probe

3.2. Optimization for the volume of probes

To obtain optimal sensitivity, we examined the influence of the detection (CRP and IL-6) probes on the microarray’s sensitivity. Different volumes of the detection probes were added to react with the same concentration of antigens; the experiment was repeated three times. The experimental results in Fig.3a show that the intensity of signal have the positive relationship with the quantity of detection probes. When adding 1 L CRP detection probe and 3 L IL-6 detection probe, the signal reached

the maximum with the background signal remaining invariable, suggesting the optimal volumes for CRP detection probe and IL-6 detection probe as 1 L and 3 L, respectively. To verify the efficiency of the signal amplification probe, we added different volumes of the probe into the assay. Figure 3b shows obvious reinforcing of the signal upon adding the signal amplification probe. The assay achieved the strongest signal at 3 L of signal amplification probe with no obvious difference in the background signal in presence of different volumes of the signal amplification probe. The signal almost enhanced by three-times with the addition of signal amplification probe. This confirms that the double AuNPs based probes (detection probe and amplification probe) can improve the sensitivity of the protein microarray. In summary, the optimal volumes for different probes to achieve maximum signal output from the assay are 1 L, 3 L, 3 L, respectively, for CRP detection probe, IL-6 detection probe, and signal amplification probe.

3.4. Optimization of incubation time and gold deposition time

In general, the intensity of signal improving with increase of incubation time. To examine the effect of incubation time on our immunoassay, we selected different incubation times from 0 to 60 min and found different signal outputs with different incubation time while keeping the detection probe, signal amplification probe and antigens same (Fig. 3c). The signal reached maximum at incubation time of 40 min with the background signal remaining almost unchanged no matter what incubation time was. Hence, 40 min was the optimum incubation time for our assay (the error bars were derived from the data of three groups).

Gold deposition were used to amplify the output signal of immunoassay. Gold deposition has an advantage of autonucleation even after 1-2 h, which improves the signal-to-noise ratio and demonstrates better signal amplification compared with silver enhancement (commonly used in signal amplification for protein detection [40]). The intensity of signal increasing sharply with gold deposition time. The signal became stable at 8 min; However, the background signal did not change with increase in gold deposition time. Therefore, to achieve maximum sensitivity for our immunoassay, the optimum time of gold deposition is 8 min. (the error bars were derived from the data of three groups).

Figure 3. (a) Volume optimization for the detection probes, (b) volume optimization for the signal amplification probe, (c) optimization of incubation time (d) optimization of gold deposition time.

3.5. Specificity and selectivity of detection

To verify specificity, we challenged the immunoassay with several possible interfering proteins such as tumor necrosis factor-α (TNF-α), myoglobin (MYO), troponin (cTnI), human serum albumin (HSA), and bovine serum albumin (BSA); PBS was used as the control. The concentration of above proteins was 100 ng/mL. In Figure 4a, CRP and IL-6 causing a tremendous increase in signal intensity , while the signal intensities for TNF-α, MYO, cTnI and HSA were roughly the same with the control-PBS. These results suggest the excellent specificity of our sandwich assay towards CRP and IL-6. To verify “no cross-reaction” among CRP antibodies, IL-6 antibodies and antigens, we mixed the double AuNPs probes with PBS, CRP, and IL-6, respectively. We observed no signal while adding PBS (Fig.4b), indicating no cross reaction among CRP antibodies and IL-6 antibodies. When we added CRP or IL-6 individually into the assay, CRP antibodies just captured CRP only, while IL-6 antibodies captured only IL-6 (Figures 4c, 4d) i.e., no cross reaction occurred either between CRP antibody and IL-6 or between IL-6 antibody and CRP. Hence, no cross reaction occurred either between antibodies and antibodies or between antibodies and antigens.

Figure 4. (a) Specificity test with different antigen, (b) only PBS, (c) only CRP, (d) only IL-6.

3.6 Sensitivity of the immunoassay towards CRP and IL-6 detection

We evaluated the analytical potential of this multiple-target platform under the optimal experimental conditions. The concentrations of antigens used were 100, 50, 25, 12.5, 9, 6.25, 5, 3.125, 2.5, 1.56, 0.78, 0.39, 0 ng/mL; each assay repeated three times. Nonlinear relationships between signal intensity and the concentration of target molecules were showed in Figure 5. Limit of detection (LOD) for CRP was estimated to be 326 pg/mL [(y = 1006.36－912.57/ (1+(x/4.74)2.77), R2 = 0.996)], and limit of detection (LOD) for IL-6 was estimated to be 8 pg/mL [(y = 1004.13－910.78/ (1+(x/1.51)1.30), R2 = 0.991)]. The sensitivity was almost invariant when compared with traditional ELISA.

Figure 5. Calibration curves for the detection of CRP and IL-6; error bars were obtained from three parallel experiments. Concentration are by log2 transformation.

3.7. Detection of real samples

To verify the clinical applicability, 28 clinical serum samples were detected by the newly developed immunoassay and a traditional ELISA kit. Figure 6 shows the uniformity in the results obtained from using the above two different assays. The X-axis represents the concentration measured by ELISA kit, while the y-axis represents the concentration measured by our immunoassay. We obtained R2 of 0.986 and 0.997, respectively, for CRP and IL-6 and a good linear relationship between ELISA kit and our microarray. The results obtained for clinical serum samples using the two different assays are almost the same, suggesting the potential of our immunoassay for detecting CRP and IL-6 simultaneously in clinical serum samples.

Figure 6. Correlation of the concentrations of clinical serum samples measured by ELSA kit and the new scanometric immunoassay. (a) CRP, (b) IL-6.

4. Conclusions

In summary, we have built a colorimetric immunoassay platform based on dual signal amplification for sensitive multiple-targeted CVD protein biomarkers detection. Double AuNPs probes and gold depositions are applied as a signal increasing mechanism for this robust, simple, and high signal-to-noise ratio assay. The LOD of picogram per microliter level are successfully achieved with two model analytes CRP (326 pg/mL) and IL-6 (8 pg/mL). Results further uncover that high specificity of this multiple immunoassay has been evaluated in the several interfering proteins mixed serum. More importantly, the simultaneous and accurate detection of CRP and IL-6 in clinical serum samples, indicating its enormous potential for clinical application. Ultimately, although this work focused on the detection of protein CVD biomarkers, this multiplexing immunoassay based on dual signal amplification could be utilized to detect various biomarkers in broader clinical samples (such as oral fluid and urine).

Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant number: 61571077, 61571429, 61801464 and 61801465), National Key Research and Development Program of China (No. 2018YFA0108202 and 2017YFA0205300), and the Science and Technology Commission of Shanghai Municipality (16410711800 and 17JC1401001).

Conflict of Interest

The authors declare no conflicts of interest.

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