Detection Of 5-Hydroxymethylfurfural And Β-Amyloid Peptide Aggregates By Quartz Crystal Microbalance Based On Gold Nanoparticle Signal Amplification

With the development of technology and the improvement of living standards,people’s demand for quality of life has gradually increased,especially the issue of food safety has become the focus of most people’s attention.Recently,most food products have gone through processing steps in the pursuit of taste,and with it comes a multitude of food contaminants.5-Hydroxymethylfurfural（HMF）is produced from reducing sugars or amino acids in foodstuffs during heating or fermentation by the Millard or caramelization reaction,and some brown substances and flavor substances are produced at the same time.So,HMF can be used as a performance indicator of the degree of food processing in the food processing process.However,it has been found that high concentrations of HMF can cause some damage to eyes and mucous membranes and to be cytotoxicity,and it can be further transformed to produce 5-sulfomethylfurfural（SMF）and 5-chloromethylfurfural（CMF）,which are clearly cytotoxic and carcinogenic.Alzheimer’s disease（AD）is a common neurodegenerative disease of the elderly that causes symptoms such as mental deterioration and memory impairment,there is no better medical treatment for it.β-amyloid peptide（Aβ）is formed through sequential cleavage of amyloid precursor protein（APP）by the proteolytic action ofβ-andγ-secretase.Aβaggregates including soluble oligomer（AβO）as well as insoluble fibril（AβF）,which are highly neurotoxic in the cellular matrix.Studies have shown that the amount of Aβaggregates in the cerebrospinal fluid（CSF）has a strong correlation with the severity of AD.Therefore,Aβaggregates can be used as a major marker of AD.Since HMF and Aβ₄₀ aggregates as two substances that have a significant impact on human health,it is urgent to develop a rapid and sensitive method to detect them.Quartz Crystal Microbalance（QCM）is a real-time online mass sensing platform,which is capable of detecting nanomolar level mass changes on the surface of quartz crystal electrodes.In this work,we have constructed two QCM biosensing platforms to detect HMF and Aβ₄₀ aggregates.（1）A QCM biosensing platform for ultrasensitive detection of HMF was constructed based on split-DNAzyme assisted AuNPs signal amplification.DNAzyme is a type of metal-dependent single stranded DNA sequence,which can recognize and cleave phosphodiester bond of individual ribonucleotide on DNA chain in the presence of specific metal ions.In this work,we designed the split-DNAzyme based on DNAzyme,containing two fragments of DNAzyme,an amino-modified strand（ESⅠ）and an aldehyde-modified strand（ESⅡ）,which can be reconstituted into a complete DNAzyme by Schiff base condensation reaction.The study showed that the reconstituted DNAzyme activity was almost unchanged,and could still recognize and cleave the corresponding sites of the AuNPs-ssDNA under the action of Mg²⁺.Due to the small molecular weight of HMF,the aldehyde group of its molecule structure can preferentially react with ESⅠ,thereby preventing the recombination of DNAzyme and further reducing the cleavage of AuNPs-ssDNA,thus establishing a correspondence between the content of HMF and AuNPs-ssDNA.The capture DNA is capable of complementary hybridization with ssDNA,so that as the HMF content increases,the AuNPs-ssDNA content also gradually increases,resulting in a large frequency change in the QCM.The detection range of the QCM biosensor for HMF was 5 nM～5μM and the detection limit was 1.24 nM,which was significantly lower than other methods.（2）A simple and rapid QCM biosensing platform for the detection of Aβ₄₀aggregates has been constructed based on the specific response of the aptamer and Aβ₄₀aggregates.Aptamer is a type of oligonucleotide sequence that can binds to a target molecule with extremely high affinity and specificity.It was found that Aβ₄₀ has two aptamer sequences（Apt1 and Apt2）and its oligomer(Aβ₄₀O)and fibril(Aβ₄₀F)can be recognized and bound by both two aptamers simultaneously,but not the monomer.On this basis,a QCM biosensing platform was constructed to detect Aβ₄₀O and Aβ₄₀F in a one-step method.Apt2 was modified on the surface of the Auchip via Au-S bond,then a mixture of the target and AuNPs-Apt1 was injected into the QCM chamber for reaction,where Apt2 specifically recognized and bound Aβ₄₀O or Aβ₄₀F,capturing it on the surface of the Auchip,the response signal was amplified due to the presence of AuNPs.The sensing platform were able to detect Aβ₄₀O in the range of 0.2～10 pM with a detection limit of 0.11 pM and Aβ₄₀F in the linear range of 0.1～10 pM with a detection limit of 0.02 pM.This method is simple and sensitive.