A wireless magnetoelastic DNA-biosensor amplified by AuNPs for the detection of a common mutated DNA causing β-thalassaemia

Highlights

• An ME DNA-biosensor was developed to detect a common mutated DNA causing β-thalassaemia.

• sDNA-AuNPs serve as the signal amplifier.

• The sandwich-type structure enables label-free detection.

• A low detection limit of 0.571 pM and high sensitivity of 72.7 Hz/nM were obtained.

• The DNA-biosensor exhibits excellent sensitivity, selectivity and stability.

Abstract

β-Thalassaemia is an inherited blood disorder with serious complications. Combining the unique advantages of DNA and magnetoelastic (ME) materials, an ME DNA-biosensor was proposed that can wirelessly detect a target DNA sequence (tDNA) that is a 4-bp deletion in codon 41/42 (-TTCT) in the β-globin gene causing β-thalassaemia. The thiolated capture probe (CP) was covalently immobilized on the surface of the gold-plated ME chip and then hybridized to tDNA, and AuNPs modified with thiolated signal probe DNA (sDNA-AuNPs) serve as the signal amplifier and the direct signal indicator, enabling label-free detection. The specific hybridization process of sDNA-AuNPs to tDNA increased the surface load mass and decreased the resonance frequency of the DNA-biosensor. The resonance frequency shift of the DNA-biosensor was linear to the logarithmic concentration of tDNA in the range of 1.0 × 10⁻⁸ M to 1.0 × 10⁻¹² M, with a detection limit (LOD) of 0.571 pM (S/N = 3) and sensitivity of 72.7 Hz/nM. The ME DNA-biosensor exhibits excellent selectivity and stability for the detection of the mutated DNA (4-bp deletion in codon 41/42) causing β-thalassaemia, suggesting this is a promising method for the clinical diagnosis of β-thalassaemia.

Introduction

β-Thalassaemia is a monogenic hereditary disorder with a high incidence rate [1,2]. It is prevalent worldwide, including Africa, Southeast Asia, the Indian subcontinent, northern Europe, North and South America, the Caribbean, and Australia, affecting the health of more than 5 % of the world's population [[3], [4], [5]]. β-Thalassaemia is caused by mutations in the β globin gene that lead to a decrease or no synthesis of a normal β-globin chain. The defect in β chain synthesis leads to an imbalance of α and β globin, resulting in the ineffective formation of red blood cells [6,7]. More than 200 mutations in β-thalassaemia have been reported worldwide, including the 4-base deletion (-TTCT) at the 41/42 position of the β-globin coding region codon, which is the most common pathogenic mutation and was selected as the target DNA sequence (tDNA) for β-thalassaemia determination in this work [8]. Current common screening methods for β-thalassaemia include polymerase chain reaction (PCR) assay [9], complete blood count (CBC) [10], allele-specific oligonucleotide (ASO) hybridization after DNA amplification [11], and amplification of refractory mutation systems (ARMS) [12]. However, these methods are costly, time consuming, difficult to perform, and even inaccurate, so there is an urgent need to develop improved diagnostic methods of β-thalassaemia [8].

Due to advantages of wireless monitoring, small size, fast response, high sensitivity, and low cost, mass-sensitive magnetoelastic (ME) biosensors made of ferromagnetic metallic glass ribbon, have attracted significant attention in the biomedical field [13,14]. ME biosensors have been widely used to successfully detect various substances, such as Salmonella [15,16], E. coli [17], and glucose in urine samples [18]. Based on the magnetostrictive effect of the ME material, ME sensors generate vibration in an applied alternating magnetic field provided by the energized coil, during which magnetic energy is converted into mechanical energy. When its vibration frequency coincides with the frequency of the applied magnetic field, the biosensor exhibits a physical resonance. The fundamental resonance frequency of the ME chip is given by the following Eq. (1) [19]:

$$f=\frac{1}{2L}\sqrt{\frac{E}{\rho (1-{\nu }^2)}}$$

Where L, E, ρ, and ν are the length, elastic module, density, and Poisson's ratio of the sensor chip, respectively. When the relevant biomolecules are captured on the ME biosensor surface, the mass loading of the ME biosensor increases and the resonance frequency decreases, as described by equation (2) [20]:

$$\Delta f=-\frac{f}{2}\frac{\Delta m}{M}$$

Where M is the initial mass of the biosensor and Δm (Δm<<M) is the surface mass change of the biosensor.

Because the quality of the DNA molecule is relatively small, it is necessary to find an appropriate operation to amplify the signal to improve the sensitivity of the biosensor. One successful approach is to introduce the amplification strategy by nanomaterials with excellent properties such as low toxicity, high biocompatibility, strong fluorescence, and high catalytic activity [[21], [22], [23]].

Based on the advantages of high chemical stability, good biocompatibility and high specific surface area [[24], [25], [26], [27]], AuNPs modified with thiolated signal probe DNA (sDNA-AuNPs) through Au-S bonds were used for signal amplification in this work. DNA biosensors typically have high physicochemical stability, and the use of magnetoelastic (ME) materials as sensing platforms provide advantages of low cost, small size, fast response, and, importantly, wireless transmission. Here we design a novel ME DNA-biosensor for the wireless detection of the mutated DNA sequence of the 4-bp deletion (–TTCT) causing β-thalassaemia. As shown in Scheme 1, the thiolated DNA that is semi-complementary to tDNA is used as the capture probe (CP), which is modified on the gold-coated ME material surface by Au-S bonds. At the same time, 6-mercapto-1-hexanol (MCH) is used to reduce non-specific adsorption. Then, tDNA is bound to the ME material surface by DNA hybridization. Finally, sDNA-AuNPs that are semi-complementary to tDNA are not only used as direct signal indicators enabling label-free detection, but are also used for signal amplification. Combined with the signal amplification strategy enhanced the sensitivity, the ME DNA-biosensor provides a potential tool for β-thalassaemia detection.