SERS “hot spot” enhance-array assay for misfolded SOD1 correlated with white matter lesions and aging

Abstract

Misfolding of superoxide dismutase-1 (SOD1) has been correlated with many neurodegenerative diseases, such as Amyotrophic lateral sclerosis's and Alzheimer's among others. However, it is unclear whether misfolded SOD1 plays a role in another neurodegenerative disease of white matter lesions (WMLs). In this study, a sensitive and specific method based on SERS technique was proposed for quantitative detection of misfolded SOD1 content in WMLs. To fabricate the double antibodysandwich substrates for SERS detection, gold nanostars modified with capture antibody were immobilized on glass substrates to prepare active SERS substrates, and then SERS probes conjugated with a Raman reporter and a specific target antibody were coupled with active SERS substrates. This SERS substrates had been employed for quantitative detection of misfolded SOD1 levels in WMLs and exhibited excellent stability, reliability, and accuracy. Moreover, experimental results indicated that the level of misfolded SOD1 increased with the increase in age and the degree of WMLs. Hence, misfolded SOD1 may be a potential blood marker for WMLs and aging. Meanwhile, SERS-based gold nanostars have great clinical application potential in the screening, diagnosis and treatment of WMLs.

Introduction

The white matter lesions (WMLs), also named as leukoaraiosis (LA) or white matter hyperintensities (WMHs), are often found in aging brain. As a surrogate of cerebral small vessel disease (SVD), the change of white matter in WMLs, encamping periventricular WMLs and deep/subcortical WMLs, is characterized by high-intensity signal abnormalities on T2-weighted magnetic resonance imaging (T2-MRI) or fluid-attenuated inversion recovery (FLAIR) images [[1], [2], [3]]. Previous research has shown that the incidence and diagnosis rate of WMLs in ageing cohort are increasing annually [4]. In the elderly population, cerebral WMLs frequently occur at the age of 60–70, but the prevalence of WMLs is near to 100% at the age of 80–90 [5]. Hence, age seems to be one of the primary risk factors for the formation of WMLs [6]. Besides, other research suggests that the oxidative stress, which is produced by hypoxia and ischemia, also plays a key role in the pathogenesis of WMLs [[7], [8], [9]].

SOD1 is an antioxidant defense protein which can metabolize superoxide radicals and catalyze the dismutation of superoxide to hydrogen peroxide and oxygen [10]. In addition to its free radical scavenging capacity, SOD1 also has a toxic-gain-of-effect. This enhanced cytotoxic effect of SOD1 is driven by the fact that both conformational mutant SOD1 and wild-type SOD1 are prone to misfolding in comformation [11,12]. Numerous studies have demonstrated that some common molecular pathologies, like as misfolding, aberrant accumulation and malfunctioning of disease-related proteins, exist in various neurodegenerative diseases [[13], [14], [15]]. Likewise, misfolded wild-type SOD1 as an abnormal proteins is often discovered in various degenerative disorders such as amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD), which display the same pathological structural conformations [13,16]. Regarding the conformational misfolding of SOD1, oxidative stress is considered to be the most common way. Experimental evidence has demonstrated that the production of misfolded SOD1 is closely related to the endoplasmic reticulum (ER) and mitochondria [17,18], particularly in the nervous system. The ER and mitochondria are the most significant sites for oxidative stress and misfolded SOD1 is usually enriched in the aggregates region for mitochondria [17]. Aging-associated oxidative stress is more relevant during SOD1 post-translational modifications to form its misfolded structural conformation [19]. Non-native alterations of SOD1 caused by oxidative stress is regarded to be involved in accelerated course or pathological aging in the setting of ALS, AD, and PD [20]. Some researchers have discovered that in ALS, the accumulation of this misfolded conformer protein usually appears in the motor neuron when pathological aging occurs [21]. In studies for AD neurotoxic mechanisms, a large quantity of SOD1 aggregated in substantia nigra (SN) and locus coeruleus (LC), suggesting that SOD1 is involvement of the pathological aging for PD [22], During the process of pathological aging of AD, misfolded SOD1 is able to trigger amyloid-β (Aβ) aggregation, which plays a key role in the initiation and development of Alzheimer's and its cognitive defects, to form plaque in intracellular [20]. Earlier, it is also demonstrated that the WMLs were associated with the aging-related degenerative diseases. For example, WMLs and AD have certain pathological alterations in common [23,24]. 70-year-old patients who was diagnosed with AD have some degree of WMLs, according to the study of mixed pathologies [24]. Similarly, some researches also have demonstrated that WMLs were the most important pathogenic factors contributing to vascular parkinsonism [25]. However, whether aging and misfolded SOD1 acts a crucial role in white matter lesions (WMLs) pathogenesis is uncertain.

In the past few years, surface-enhanced Raman scattering (SERS) as a highly sensitive spectroscopic technique has been introduced in many fields, including food safety monitoring, environmental sensing, chemical analysis and medical diagnostics [[26], [27], [28]]. The mechanism of Raman signals amplification can be attributed to the dramatically enhanced electromagnetic field in “hot spots” regions created by the overlapping of multiple metallic nanoparticles or vertices in single nanostructures, As a result, the enhancement of Raman signals can reach 8 to 10 orders of magnitude [[29], [30], [31]]. Compared with other detection methods, there are some distinct advantages for SERS. For example, SERS measurement does not require complex samples, and its operation procedure is simple when used for sample analysis immediately. Moreover, it can detect multiple bio-molecules in a single sample simultaneously with high specificity because of its narrow bandwidth of fingerprints and less spectral overlap [[32], [33], [34]]. Besides, SERS signals can become more reproducible, stable and powerful when specific metal materials (Au and Ag) are selected as Raman enhancing platforms which provide long-term stability, high biocompatibility and the specific nanostructure [35,36]. Among many specific nanostructures, gold nanostars (GNSs) are capable of acting as effective amplifiers due to multi-branched structure which can create a huge number of hot spots. This nanomaterial has recently been introduced in screening and diagnosis of many diseases, such as infectious diseases [37], chronic diseases [38] and tumours [39]. Nonetheless, SERS sensors based on GNSs are very rare to find reports on the quantitative detection of misfolded SOD1 levels in WMLs. Meanwhile, few studies have been conducted on the relationship between misfolded SOD1 and WMLs.

Herein, a highly sensitive SERS sensors based on GNSs was utilized to detect the concentration of misfolded SOD1 in the WMLs patients’ blood for the first time. And then the correlation among the misfolded SOD1 and the severity of WMLs as well as aging was explored (Scheme 1). Firstly, the multibranched structure of GNSs with greatly enhanced SERS intensity was synthesized by seed-mediated growth method [27]. Then, the special antibody and Raman reporters were modified onto gold nanoparticle surfaces to form SERS probes by employing covalent bonding [40]. In order to generate high-performance SERS biosensors, the gold nanostars were uniformly immobilized on the treated glass slide by the spin coating technique. Anti-misfolded SOD1 and Raman reporters were modified onto gold nanoparticle surfaces to form SERS probes. Finally, the SERS probes were attached to glass substrates to assemble a sandwich-type SERS biosensor plate. The feasibility of this method was verified by enzyme linked immunosorbent assay (ELISA) experiment. Thereby, it can be provided for ultrasensitive qualitative and quantitative detection of misfolded SOD1 in blood sample. The relation of the misfolded SOD1, white matte lesions and aging was analyzed by using univariate and multiple logistic regression analyses. In this work, a new approach for the first quantitative SERS-based analysis of the correlation between misfolded SOD1 and WMLs as well as age is presented, implying the potential value of the highly sensitive, reproducible, and quick detection method and misfolded SOD1 for closer monitoring and diagnosis of white matte lesions and aging.