Sandwich-like electrochemical aptasensing of heat shock protein 70 kDa (HSP70): Application in diagnosis/prognosis of coronavirus disease 2019 (COVID-19)

Abstract

In this research, by using aptamer-conjugated gold nanoparticles (aptamer-AuNPs) and a modified glassy carbon electrode (GCE) with reduced graphene oxide (rGO) and Acropora-like gold (ALG) nanostructure, a sandwich-like system provided for sensitive detection of heat shock protein 70 kDa (HSP70), which applied as a functional biomarker in diagnosis/prognosis of COVID-19. Initially, the surface of the GCE was improved with rGO and ALG nanostructures, respectively. Then, an aptamer sequence as the first part of the bioreceptor was covalently bound on the surface of the GCE/rGO/ALG nanostructures. After adding the analyte, the second part of the bioreceptor (aptamer-AuNPs) was immobilized on the electrode surface to improve the diagnostic performance. The designed aptasensor detected HSP70 in a wide linear range, from 5 pg mL^-1 to 75 ng mL^-1, with a limit of detection (LOD) of ∼2 pg mL^-1. The aptasensor was stable for 3 weeks and applicable in detecting 40 real plasma samples of COVID-19 patients. The diagnostic sensitivity and specificity were 90% and 85%, respectively, compared with the reverse transcription-polymerase chain reaction (RT-PCR) method.

Keywords: Aptasensor; Bioelectrochemistry; Coronavirus disease 2019 (COVID-19); Gold nanomaterials; Heat shock protein 70 kDa (HSP70); Reduced graphene oxide (rGO).

Fig. 1

Schematic presentation of the sandwich-like aptasensing platform to diagnose COVID-19 through detection of HSP70.

Fig. 2

FESEM micrographs of GCE (a), GCE/rGO (b), GCE/Gold nanostructure: electrodeposition time: 400 S, magnification 30 K (c), GCE/Gold nanostructure: electrodeposition time: 400 S, magnification 40 K (d), GCE/Gold nanostructure: electrodeposition time: 250 S, magnification 10 K (e), GCE/Gold nanostructure: electrodeposition time: 250 S, magnification 15 K (f), GCE/rGO/ALG nanostructure: electrodeposition time: 250 S, magnification 10 K (g), GCE/rGO/ALG nanostructure: deposition time: 250 S, magnification 15 K (h), UV–Vis of AuNPs and aptamer-AuNPs (i), TEM micrographs of AuNPs (j), and aptamer-AuNPs (k). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

CVs related to the various states of the signal transducer (scan rate: 200 mV S⁻¹) (a); EIS analysis of the various states of the signal transducer: a) GCE, b) GCE/rGO, c) GCE/rGO/ALG nanostructure, d) GCE/rGO/ALG nanostructure/aptamer (without analyte), e) GCE/rGO/ALG nanostructure/aptamer/HSP70 (1 ng mL⁻¹)/aptamer-AuNPs; DPVs of the various states of the signal transducer (c); DPVs (signal-off) of aptasensor in the presence of various concentrations of HSP70 (d); calibration curve of HSP70 aptasensor (Ip vs. logarithm of analyte concentrations) (e); Error bars indicate standard deviations from three repeated measurements (error bar: SD/n = 3).

Fig. 4

Reproducibility performance for seven consecutive repeated refabricating of aptasensor (a); stability of aptasensor for 21 days analysis (b); specificity analysis of aptasensor in the presence of HSP90, Hb, heparin, HSA, IgG, and mixtures of all interfering agents; Error bars indicate standard deviations from three repeated measurements (error bar: SD/n = 3).