In situ enzymatic generation of gold for ultrasensitive amperometric sandwich immunoassay of procalcitonin
Introduction
Procalcitonin (PCT) is a protein produced by the C cells of the thyroid gland. The serum concentration of PCT in healthy human is as low as 0.1 ng mL−1 (Liu et al., 2014), and its level can be somewhat elevated after suffering from sepsis, multiple organ failure or serious infection of bacteria, fungi, or parasites (Li et al., 2014, Liu and Wang, 2015). PCT is a valuable indicator for clinical diagnosis, treatment and prognosis of the bacterial inflammatory diseases (Yuan et al., 2016). Immunoassay is the common method for the serum PCT assay, and the conventional immunoassay methods for clinical serum PCT detection (e.g. radioactive immunoassay and enzyme-linked fluorescent assay) still face some challenges in sensitivity, safety, cost and/or operational convenience. Hence, studying and developing new and ultrasensitive methods for trace analysis of PCT is of sufficient bioanalysis and biomedical significance.
Usually, the immunoassay relies on the immunolabelling methods to output and amplify the analytical signal, and the reported immunolabelling methods can be divided into two classes, molecule/atom-based and nanomaterial-based immunolabelling, because immunolabelling at molecule/atom size and nanoscale can well match the molecular sizes of antigen and antibody for signal readout. Both the molecules/atoms and the nanomaterials employed for immunoassay are functional reagents/materials for a convenient signal readout, e.g. catalytic enzyme molecules, radioactive atoms and ambient-stable metal nanomaterials. Metal immunoassay selectively converts the specific immunoaffinity event into a readable metal signal and has the advantages of high sensitivity, favorable simplicity and low cost of instrumentation (Ambrosi et al., 2010, Dequaire et al., 2000). Anodic stripping voltammetry (ASV) as a highly sensitive and convenient electrochemical method is usually employed for metal quantification in metal immunoassay. In a conventional metal immunoassay, metal nanomaterials are directly employed to label the second antibody, and either the detection of this metal label or the detection of the second metal after its selective chemical staining on the metal label yields the immunoassay signal (Chen et al., 2007, Lai et al., 2011). The signaling metal can also be generated by an appropriate enzyme label, and the enzyme-catalytic metal deposition offers the extremely high specificity and efficiency (Chen et al., 2007, Lai et al., 2011). Hence, the metal immunoassay in combination with enzyme labelling is a very promising way to achieve high sensitivity. However, the ASV detection of the metal in metal immunoassay is generally conducted at a fresh working electrode in a milliliter-scale solution diluted from the metal lysate, thus the solution dilution must lower the immunoassay sensitivity (Qin et al., 2015). To overcome the sensitivity barrier, we have recently introduced in-situ microliter-droplet ASV directly on the immunoelectrode for metal immunoassay of improved sensitivity (Qin et al., 2015, Qin et al., 2016, Qin et al., 2017), which appears to be a promising protocol worthy of further researches for ultrasensitive metal immunoassay and metal-labeled bioanalysis of many other analytes. In addition, Au nanomaterials are widely used in metal immunoassay and amperometric bioanalysis, but the reported dissolution of Au by HBr-Br2 for ASV analysis of Au still leads to some problems such as background interferences (Dequaire et al., 2000, Qin et al., 2015).
Herein, on the basis of glucose oxidase (GOx)-catalyzed Au deposition and in-situ microliter-droplet ASV of the enzyme-generated Au directly on the immunoelectrode, we report an ultrasensitive amperometric sandwich immunoassay of PCT. Under optimized conditions, the limit of detection (LOD, S/N = 3) of PCT is as low as 0.04 fg mL−1, which is much lower than those reported for PCT (Table S1). In addition, a new method of dissolving Au by an appropriately diluted aqua regia is suggested, which can yield an ASV peak of Au with improved resolution compared to the reported protocol of dissolving Au by HBr-Br2 (Dequaire et al., 2000, Qin et al., 2015).
Section snippets
Materials and reagents
Recombinant human procalcitonin (PCT, MW ≈ 13 kDa) and mouse anti-human procalcitonin (anti-PCT) were donated by College of Medicine of Hunan Normal University. GOx (EC 1.1.3.4; type II from Aspergillus niger, activity≈10 kU g−1, MW ≈ 154 kDa), bovine serum albumin (BSA), chloroauric acid (HAuCl4), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich. L-DOPA was purchased from Aladdin. NaAuCl4 solution was obtained
Characterization of the materials and immunoelectrodes
The UV−Vis spectrophotometric studies are shown in Fig. 2. NaSCN and glucose solutions were colorless (samples 1 and 3) with no significant absorption peak in the measured wavelength range. The color of BQ solution was in light pink (sample 2) with an absorption peak at 421 nm that is the characteristic absorption peak for BQ (Gu et al., 2017). The HAuCl4 solution was in light yellow, the Au bio-staining solution was in light pink, and both solutions showed some UV–Vis absorption signals in the
Conclusions
In conclusion, the ultrasensitive sandwich immunological electrochemical detection of PCT is achieved on the basis of GOx-catalyzed gold staining and in-situ microliter-droplet ASV of the enzyme-generated gold directly on the immunoelectrode. A new method of dissolving gold by an appropriately diluted aqua regia and the simultaneous cathodic preconcentration of gold on the immunoelectrode is demonstrated for better conducting ASV analysis of gold than the reported one. Under optimized
Acknowledgments
This work was supported by the National Natural Science Foundation of China (21475041, 21675050, 20405142, 21305041), Hunan Lotus Scholars Program (2011), and Foundation of the Science & Technology Department of Hunan Province (2016SK2020).
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