Determination of Alzheimer biomarker DNA by using an electrode modified with in-situ precipitated molybdophosphate catalyzed by alkaline phosphatase-encapsulated DNA hydrogel and target recycling amplification

An electrochemical biosensor is described for highly sensitive determination of tDNA, an Alzheimer’s disease (AD)-related biomarker. Electroactive molybdophosphate anions were precipitated in-situ on a glassy carbon electrode (GCE) via catalytic hydrolysis by alkaline phosphatase (ALP). This is followed by recycling amplification of tDNA. Four DNA strands (referred to as S1, S2, S3 and S4) were designed to assemble X-shape DNA (X-DNA) building blocks. These were further extended into four directions under the action of DNA polymerase. The resultant two X-DNA motifs were polymerize. Simultaneously, ALP is encapsulated into a hydrogels network to obtain a porous material of type ALP@DNAhg. The GCE was modified with reduced graphene oxide functionalized with gold nanoparticles (Au@rGO). If ALP@DNAhg are captured via strand displacement, tDNA recycling assembly for signal amplification is initiated. This results in the immobilization of large amounts of ALP. On introduction of pyrophosphate and molybdate (MoO 4 2− ), ALP will catalyze the hydrolysis of pyrophosphate to produce phosphate. It will react with molybdate to form redox active phosphomolybdate anions (PMo 12 O 40 3− ). Its amperometrical signal depends on the concentration of tDNA in the 1.0 × 10 −2 to 1.0 × 10 4 pM concentration range, and the detection limit is 3.4 × 10 −3 pM. Graphical abstract Schematic presentation of ( a ) preparation of alkaline phosphatase-encapsulated DNA hydrogel (ALP@DNAhg). ( b ) fabrication of the biosensor for target DNA (tDNA) based on ALP@DNAhg to catalyze in situ precipitation of electroactive molybdophosphate anion (PMo 12 O 40 3− ) and tDNA recycling amplification, achieving tDNA-dependent electrochemical signal readout (X-DNA: X-shape DNA building block. TdT: terminal deoxynucleotidyl transferase. dATP: deoxyadenosine triphosphate. dTTP: deoxythymidine triphosphate. X-DNA-pAn and X-DNA-pTn: X-DNA motifs with poly-A and poly-T tails. ALP: alkaline phosphatase. ALP@DNAhg: ALP-encapsulated DNA hydrogels. Au@rGO: gold nanoparticles-functionalized reduced graphene oxide. GCE: glass carbon electrode. HP1, 2: hairpin DNA 1, 2. MCH: 6-mercaptohexanol. tDNA: target DNA. CV: cyclic voltammetry).