An ultrasensitive electrochemical immunosensor for procalcitonin detection based on the gold nanoparticles-enhanced tyramide signal amplification strategy

Highlights

• The AuNPs-TSA immunosensor is an effective signal amplification system based on the two nanocomposites.

• AuNPs-TSA immunosensor has been developed for detection of PCT and the LOD for PCT is 1 pg mL⁻¹.

• This immunosensor could be promisingly used for clinical early diagnosis of bacterial infections and also for guiding antibiotic therapy due to its ability for highly sensitivity detection.

Abstract

In this study, we established an ultrasensitive electrochemical immunosensor based on the gold nanoparticles-enhanced tyramide signal amplification (AuNPs-TSA) for the detection of procalcitonin (PCT, for discriminating bacterial infections from nonbacterial infections). Firstly, a facilely prepared, well-conducting reduced graphene oxide nanosheets/GNP (rGO-AuNPs) nanocomposite was synthesized and immobilized on the electrode surface to absorb more capture antibodies (Ab1). Next another nanocomposite, acting as a signal tool, was modified with detection antibody (Ab2) and horseradish peroxidase (HRP), and then backfilled by bovine serum albumin (BSA). Because a single AuNP is able to load multiple HRPs and BSAs, a number of tyramine labeled biotins (T-B) could be deposited on the proteins adhering to the surface of AuNPs. Moreover, the high affinity between streptavidin (SA) and biotins significantly increases the loading of streptavidin labeled horseradish peroxidase (SA-HRP). The amplification system which was based on the two nanocomposites mentioned above, effectively amplified the electric current signals. This immunosensor exhibits a wide dynamic detection range from 0.05 ng mL⁻¹ to 100 ng mL⁻¹ and with an ultralow detection limit of 0.1 pg mL⁻¹. We have successfully utilized this immunosensor to quantify the concentration of PCT in human serum samples, and the results suggest its potential use in clinical application.

Introduction

The misuse of antibiotics not only aggravates antibiotic resistance, but also causes severe side effects on human health (Buttmann and Rieckmann, 2010). Therefore, the accurate diagnosis of infection before employing the antibiotic therapy is important and clinically necessary. Procalcitonin (PCT), a sensitive biomarker of inflammation, is a FDA-approved marker of blood infection for guiding antibiotic therapy (Schuetz et al., 2018). In clinical settings, when PCT concentration in serum exceeds 0.5 ng mL⁻¹, the antibiotic therapy will be highly recommended (Tang et al., 2007). In addition, the serum PCT level also plays an advisory role in the diagnosis of sepsis, as higher than 2 ng mL⁻¹ of PCT suggests the occurrence of sepsis (Schneider and Lam, 2007, Yu et al., 2016). Thus, the accurate detection of PCT in serum is crucial for effective early diagnosis and very helpful for further treatment guidance.

So far, numerous methods for detecting PCT based on immunoassay have been reported, including colorimetric immunoassay (Köszegi, 2002), chemiluminescence immunoassay (CMIA) (Morgenthaler et al., 2002), electrochemical immunoassay (Liu et al., 2014, Ribaut et al., 2016, Sipos and Urakawa, 2016), microfluidics immunoassays (Li et al., 2017), and fluorescence immunoassay (Chao et al., 2015). Due to the lack of signal amplification process in the detection system, these methods are either with low sensitivity or with narrow linear range. Hence, developing new methods for PCT detection are required.

Tyramide signal amplification (TSA) is a horseradish peroxidase (HRP) mediated signal amplification method. With the presence of hydrogen peroxide (H₂O₂), HRP is able to catalyze the phenolic part of tyramine to produce a radical-containing quinone-like structure on the C2 group. This “activated” tyramine then covalently binds to the tyrosine residues of nearby protein molecules (van Gijlswijk et al., 1997). Due to its good amplification effect and simple operations, TSA has been widely used in ELISA (Park et al., 2012a, Park et al., 2012b), immunohistochemistry (Wang et al., 2011), and in situ hybridization (Zubáčová et al., 2011) for the detection of protein (Akama et al., 2016), cell (Watakabe et al., 2010), and virus (Trang et al., 2015).

Herein, an ultrasensitive electrochemical immunosensor based on the gold nanoparticles-enhanced tyramide signal amplification (AuNPs-TSA) for detection of PCT has been developed. In this work, reduced graphene oxide nanosheets/GNP (rGO-AuNPs) nanocomposite was used to modify the working electrode sensing platform, to enlarge the amount of the immobilized capture antibodies (Ab1) (Chen et al., 2014). Another synthesized nanocomposite was employed as the carrier for HRP and detection antibody (Ab2). AuNPs have been reported to exhibit great chemical stability, large specific surface area, strong adsorption ability, good electrical conductivity, biocompatibility, and suitability (Chen et al., 2014, Hammond et al., 2016, Zhang et al., 2016). The use of AuNPs here could absorb more Ab2s and HRPs and meanwhile not affect the bioactivity of HRPs and Ab2s. In addition to the abovementioned advantages, HRP catalysis could also lead to the deposition of tyramine labeled biotins (T-Bs) to blocked bovine serum albumin (BSA) and hence further amplify the signal. This is not widely utilized in traditional TSA-based methods (Wu et al., 2014). This proposed immunosensor exhibits a wide linear range and an ultralow detection of limit for PCT. Most important, our results indicate that this immunosensor has good performance in human serum sample analysis, exhibiting its potential use in clinical diagnosis.