A novel electrochemical biosensor with molecularly imprinted polymers and aptamer-based sandwich assay for determining amyloid-β oligomer

Highlights

• A novel electrochemical biosensor was proposed for amyloid-β Oligomer detection.

• This sandwich assay was formed by MIPs and SiO₂@Ag-aptamer bioconjugate.

• The biosensor exhibited high sensitivity using SiO₂@Ag to amplified signal.

Abstract

Amyloid-β oligomers (AβOs) are highly toxic species involved in Alzheimer's disease (AD). Hence, a reliable detection method for AβOs, which are promising potential therapeutic targets and biomarkers for AD, is of great significance for improving the diagnosis of AD. Herein, a novel sandwich assay electrochemical biosensor was developed for highly sensitive and selective detection of AβO, using molecularly imprinted polymers (MIPs) and aptamer as the recognition element. Instead of using an antibody to recognize the AβO target molecules, the AβO in the samples were captured by the film of MIPs and the AβO-specific aptamer, forming a MIPs/target/aptamer sandwich assay for the highly selective detection of AβO. The AβO-specific aptamer was immobilized on the surface of core-shell nanoparticles that combined silver nanoparticles with silica nanoparticles (SiO₂@Ag NPs). The highly sensitive electrochemical signal from the sandwich assay was generated by using a small amount of AβO to trigger a large number of electrochemically active Ag NPs. Under the optimized conditions, the developed biosensor showed good linearity in the concentration range of 5 pg mL⁻¹ to 10 ng mL⁻¹ with a limit of detection of 1.22 pg mL⁻¹. The biosensor also showed excellent specificity, reproducibility and stability. In addition, the feasibility of detecting AβO in human serum was successfully verified, demonstrating the promising potential of this approach for clinical research and the early diagnosis of AD.

Introduction

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder, which is an enormous public health challenge due to the rapid aging of the global population [1]. The number of AD patients worldwide is estimated to reach 131.5 million by 2050, and a recent report has estimated that the annual total cost of dementia care is very high and will increase to a trillion dollars a year [2]. Thus, the early diagnosis of AD is extremely important for preventing and curing AD-caused dementia and death worldwide. According to recent reports, soluble amyloid-β oligomers (AβOs), rather than Aβ fibrils or Aβ monomers, are the most neurotoxic species involved in Alzheimer's pathogenesis [3,4]. AβOs are derived from the proteolytic cleavage of the amyloid precursor protein (APP) and obtained from Aβ peptides with lengths of 40–42 amino acids [5]. AβOs can then form the insoluble Aβ plaques that are the representative pathological feature of AD [6]. A variety of techniques have been used to detect AβOs [7], including electrochemistry [[8], [48], [49]], surface-enhanced Raman spectroscopy (SERS) [9], fluorescence [3], localized surface plasma resonance (LSPR) [[10], [45]], and mass spectrometry [11]. Although these methods have low limits of detection, they also have some inherent drawbacks, since they can be labor intensive and requiring complicated instruments and expensive antibodies. Therefore, simple, low cost, sensitive and selective methods for the early diagnosis of AD are highly desirable.

In general, AβOs can be recognized and captured by antibodies or single-chain antibody fragments [12,13]. Alternatively, molecularly imprinted polymers (MIPs) can be used as synthetic antibody mimics for specific molecular recognition, and offer the advantages of high selectivity, chemical stability, easy tailoring, resistance to harsh environments, and potential applicability to all proteins [14,15]. The template used for the synthesis of MIPs can be the target molecule or a derivative of the target molecule [16,17]. The binding sites in the imprinted cavities of MIPs bind the target molecule with excellent affinity and selectivity [18], equivalent to the performance of natural antibodies [19]. These “artificial antibodies” have received significant interest and have yielded excellent results in many applications, including separation, biosensors, catalysis, and drug delivery [16,[20], [21], [22]]. Furthermore, MIPs have the potential for extensive application in biosensing and biomarker detection.

Recently, aptamers have been considered as potential alternatives to antibodies due to their impressive recognition features and their competitive advantages [23], including easy preparation, design versatility, facile modification, low molecular weight, simple structure, and chemical stability [24]. In addition, aptamers generally have high binding affinity and high selectivity for their specific target, including metal ions [25], amino acids [26], other small organic molecules [27,28], viral proteins [[29], [30], [31]], and even entire cells [32]. Aptamers have been widely employed in the fields of diagnostics, therapeutics, molecular imaging, and biosensors [[33], [34], [35]]. In particular, biosensors based on aptamers exhibit extraordinary advantages compared with biosensors that use natural antibodies or enzymes as receptors [36]. Fortunately, Tsukakoshi's group has obtained an AβO-specific aptamer using a competitive screening method based on aptamer blotting [37], which can potentially be applied in diagnostic assays for AD.

In this study, a highly sensitive and selective electrochemical biosensor was reported for the detection of AβO using MIPs and aptamer to recognize AβO. As shown in Scheme 1A, core-shell nanoparticles that combine silver nanoparticles with silica nanoparticles (SiO₂@Ag) were introduced to generate and amplify the electrochemical signal. The AβO-specific aptamer was assembled on the surface of the SiO₂@Ag nanoparticles using a Ag-SH bond, forming a SiO₂@Ag-aptamer bioconjugate. The molecularly imprinted substrates were fabricated using a glassy carbon electrode (GCE), which was first coated with a gold nanoparticle and reduced graphene oxide (AuNPs-GO) composite, followed by a molecularly imprinted layer in the presence of the AβO template (Scheme 1B). GO and AuNPs were used to improve the electrical conductivity and the surface-to-volume ratio due to their good conductivity and high surface area [35]. The AβO in samples were then specifically captured by the MIPs film, which acted as an artificial antibody. After removing non-specifically bound species, the captured targets bound to the SiO₂@Ag-aptamer, producing electrochemical signals due to the formation of a sandwich structure on the MIPs film. This sandwich assay electrochemical biosensor showed high specificity and sensitivity towards AβO, with a limit of detection (LOD) of 1.22 pg mL⁻¹ (S/N = 3), which is similar to the concentration of AβOs in AD patients [4,8]. Additionally, this approach showed promising results in the determination of AβOs.