CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Iodine-Mediated Oxidative Annulation of ß,γ-Unsaturated Hydrazones in Dimethyl Sulfoxide: A Strategy to Build 1,6-Dihydropyridazines and Pyrroles.

Simple, commercially available iodine was successfully employed as a highly efficient and chemoselective catalyst for the oxidative annulation of ß,γ-unsaturated hydrazones to produce 1,6-dihydropyridazines under mild conditions for the first time. Interestingly, when active ß,γ-unsaturated hydrazone compounds containing electron-donating groups, such as furyl, thienyl, and cycloalkyl, were used, pyrroles were obtained. A gram-scale preparation experiment and further derivatization of pyridazines demonstrated the potential applicability of our synthesis method. Experimental studies and density functional theory calculations unveiled the origin of the chemoselectivity determining the formation of different products.

Phase separation of YAP mediated by coiled-coil domain promotes hepatoblastoma proliferation via activation of transcription.

AIM AND BACKGROUND: Yes-associated protein (YAP), a key transcriptional co-activator associated with cell fate and tumor progression, has been reported to be a powerful driver of hepatoblastoma (HB). In this study, we investigated the mechanism underlying oncogenic role of YAP in HB. METHODS: The expression of YAP in HB tissues was measured through WB and qRT-PCR. The IHC and IF were performed to determine the distribution of YAP. The phase separation of YAP was proved by living cell imaging and FRAP experiment. The effect of YAP phase separation in HB cells in vitro an in vivo were tested using CCK8, flow cytometry, and xenograft tumors. RESULTS: YAP was overexpressed and activated in HB. Nuclear YAP formed an active transcriptional site via LLPS to recruit the crucial transcription factor TEAD4. Thus, YAP phase separation facilitated transcription of oncogenic genes and subsequently mediated chemoresistance of HB. Mechanistically, the phase separation ability of YAP depends on the coiled-coil domain, which is a typical phase separation domain. The electrostatic interactions and hydrophobic interactions within YAP are also vital to YAP phase separation. More importantly, YAP inhibitor verteporfin is potential treatment for HB and combination with cisplatin enhanced therapeutic efficacy. CONCLUSIONS: Highly expressed and active YAP exerts an oncogenic effect in HB via phase separation and provides new insights for the treatment of HB.

Histone deacetylase MrHos3 negatively regulates the production of citrinin and pigments in Monascus ruber.

Monascus spp. can produce a variety of beneficial metabolites widely used in food and pharmaceutical industries. However, some Monascus species contain the complete gene cluster responsible for citrinin biosynthesis, which raises our concerns about the safety of their fermented products. In this study, the gene Mrhos3, encoding histone deacetylase (HDAC), was deleted to evaluate its effects on the production of mycotoxin (citrinin) and the edible pigments as well as the developmental process of Monascus ruber M7. The results showed that absence of Mrhos3 caused an enhancement of citrinin content by 105.1%, 82.4%, 111.9%, and 95.7% at the 5th, 7th, 9th, and 11th day, respectively. Furthermore, deletion of Mrhos3 increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. In addition, deletion of Mrhos3 led to an increase in total pigment content and six classic pigment components. Western blot results revealed that deletion of Mrhos3 could significantly elevate the acetylation level of H3K9, H4K12, H3K18, and total protein. This study provides an important insight into the effects of hos3 gene on the secondary metabolites production in filamentous fungi.

Hexamethyldisilazane Lithium (LiHMDS)-Promoted Hydroboration of Alkynes and Alkenes with Pinacolborane.

Lithium-promoted hydroboration of alkynes and alkenes using commercially available hexamethyldisilazane lithium as a precatalyst and HBpin as a hydride source has been developed. This method will be appealing for organic synthesis because of its remarkable substrate tolerance and good yields. Mechanistic studies revealed that the hydroboration proceeds through the in situ-formed BH3 species, which acts to drive the turnover of the hydroboration of alkynes and alkenes.

Electrochemical ultrasensitive detection of CYFRA21-1 using Ti3C2Tx-MXene as enhancer and covalent organic frameworks as labels.

The concentration level of cytokeratin fragment antigen 21-1 (CYFRA21-1) can be used as an important indicator for predicting non-small cell lung cancer (NSCLC). Here, a sandwich-type electrochemical immunosensor for ultrasensitive detection of CYFRA21-1 is developed. The sensor based on a combination of gold nanoparticle (AuNPs) decorated Ti3C2Tx-MXene (Au-Ti3C2Tx) as the substrate enhancer, and toluidine blue (TB) modified AuNPs doped covalent organic framework (COF) polymer as the signal tag (TB-Au-COF). The Au-Ti3C2Tx is used to capture numerous primary antibodies and accelerate the electron transfer rate of the substrate, while the TB-Au-COF can be applied to provide a large number of signal units TB and secondary antibodies. These features of composites endow the proposed immunosensor with high sensitivity and current response to CYFRA21-1. Under optimum conditions, the immunosensor offers a wide current response for CYFRA21-1 from 0.5-1.0 × 104 pg·mL-1 with a detection limit of 0.1 pg·mL-1. Furthermore, the biosensing platform can be applied for CYFRA21-1 detection to analyze real serum samples, providing an effective and useful avenue for the applicability of Au-Ti3C2Tx and TB-Au-COF composite materials in biosensing field.

Metal-Free Photoinduced Atom Transfer Radical Polymerization for Highly Sensitive Detection of Lung Cancer DNA.

Convenient and sensitive detection of biomolecules is of great significance to disease diagnosis. In this work, a metal-free photoinduced atom transfer radical polymerization (photoATRP) by a reductive quenching pathway as a novel strategy is applied to achieve lung cancer DNA detection. Thiolated PNA is exploited to specifically recognize target DNA, and the initiator of photoATRP is linked to the electrode surface via phosphate-Zr4+ -carboxylate. Under the excitation of blue light, the reductive quenching pathway is activated with eosin Y (EY) as photoredox catalyst and N,N,N',N'',N'-pentamethyldiethylenetriamine (PMDETA) as electron donor, and numerous polymeric chains are formed. Under optimal conditions, the linear range of this strategy is from 0.1 pm to 10 nm (R2 =0.989) with a limit of detection (LOD) of 1.4 fm (14 zmol in 10 µL). The variety of possible light sources for photoATRP and simple operation endow this biosensor with great potential for practical applications.

Intramolecular photoinitiator induced atom transfer radical polymerization for electrochemical DNA detection.

A novel electrochemical biosensor was reported for the first time to achieve highly sensitive DNA detection based on photoinduced atom transfer radical polymerization (photoATRP). In this work, PNA was applied as the capture probe to specifically recognize the target DNA (TDNA), and we utilized lung cancer DNA as TDNA. The ATRP initiator was introduced to the electrode surface via phosphate-Zr4+-carboxylate chemistry. PhotoATRP was activated under blue light irradiation based on a photoinitiator I2959, which produced free radicals via homolytic cleavage. Subsequently, Cu2+ was reduced to Cu+ with the assistance of the free radicals, and numerous electroactive probes were grafted onto the electrode surface. Under optimal conditions, the limit of detection (LOD) of this method was 3.16 fM (S/N = 3, R2 = 0.992), and the linear range was from 10 fM to 1.0 nM. More importantly, the preparation process of this biosensor was simple and less laborious with a low background signal, suggesting good potential in practical applications.

Analysis of Flavonoid Metabolites in Chaenomeles Petals Using UPLC-ESI-MS/MS.

Chaenomeles species are used for both ornamental decoration and medicinal purposes. In order to have a better understanding of the flavonoid profile of Chaenomeles, the petals of four Chaenomeles species, including Chaenomeles japonica (RB), Chaenomeles speciose (ZP), Chaenomeles sinensis (GP), and Chaenomeles cathayensis (MY), were selected as experimental material. The total flavonoid content of GP was found to be the highest, followed by MY, ZP, and RB. In total, 179 flavonoid metabolites (including 49 flavonols, 46 flavonoids, 19 flavone C-glycosides, 17 procyanidins, 15 anthocyanins, 10 flavanols, 10 dihydroflavonoids, 6 isoflavones, 5 dihydroflavonols, and 2 chalcones) were identified by Ultra-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry. Screening of differential flavonoid metabolites showed that GP had higher levels of metabolites when compared with the other three Chaenomeles species. Annotation and enrichment analysis of flavonoid metabolites revealed that cyanidin 3,5-diglucoside and pelargonidin-3,5-diglucoside anthocyanins are likely responsible for the color differences of the four Chaenomeles petals. Additionally, a large number of flavonoids, flavonols, and isoflavones were enriched in the petals of GP. This study provides new insights into the development and utilization of Chaenomeles petals and provides a basis for future investigations into their utilization.

Ultrasensitive Detection of DNA via SI-eRAFT and in Situ Metalization Dual-Signal Amplification.

In this work, we report a new amplification strategy based on electrochemically mediated reversible addition-fragmentation chain transfer (eRAFT) and in situ metalization for electrochemical detection of DNA. First, peptide nucleic acid (PNA) probes were immobilized on the surface of the gold electrode, and when they hybridized with the target DNA, the chain transfer agent (CTA), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD), of RAFT was connected to the PNA/DNA heteroduplex formed by the coordination bonding of Zr4+. Then glycosyloxyethyl methacrylates (GEMA) were assembled on the surface of the electrode by electrochemically mediated surface-initiated reversible addition-fragmentation chain transfer (SI-eRAFT) to form a polymer-containing sugar glucose. Next, the o-hydroxyl groups on the polysaccharide molecular skeleton were oxidized to aldehyde groups by sodium periodate (NaIO4). The aldehyde groups generated then reduce silver ions to silver particles deposited on the electrode surface in situ, and this system was then subjected to differential pulse voltammetry (DPV). Under optimal conditions, the intensity of the stripping current and the logarithm of the target DNA (tDNA) concentration has a good linear relationship in the range of 10 aM to 1 pM (R2 = 0.996), and the detection limit can go down to 5.4 aM (S/N = 3). Moreover, the method is suitable for single-nucleotide polymorphism (SNP) analysis and has strong anti-interference ability for the analysis of target ssDNA in serum samples.

Simple and fast electrochemical detection of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes with ethynylferrocene.

A universal and straightforward electrochemical biosensing strategy for the detection and identification of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes (hairpins) with ethynylferrocene was reported. In the target-unbound form, the immobilized hairpins were kept in the folded stem-loop configuration with their azido terminals held in close proximity of the electrode surface, making them difficult to be labeled with ethynylferrocene due to the remarkable steric hindrance of the densely packed hairpins. Upon hybridization, they were unfolded and underwent a large conformational change, thus enabling the azido terminals to become available for its subsequent conjugation with ethynylferrocene via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). After that, the quantitatively labeled ethynylferrocene could be exploited as the electroactive probes to monitor the DNA hybridization. As the unfolded hairpins were labeled in a stoichiometric ratio of 1 : 1, the electrochemical measurement based on differential pulse voltammetry enabled a reliable quantification of sequence-specific DNA. Under optimal conditions, the strategy could detect target single-stranded DNA (ssDNA) down to 0.296 pM with a good linear response over the range from 1 pM to 1 nM, and had excellent specificity in the genotyping of single-nucleotide polymorphisms. Furthermore, it also exhibited good detection reliability in serum samples and required no complicated protocols. More importantly, the simplicity of this strategy together with its compatibility with microfluidic chips makes it show great potential in clinical applications, where simple procedures are generally preferred.

Effective enhancement of the ability of Monascus pilosus to produce lipid-lowering compound Monacolin K via perturbation of metabolic flux and histone acetylation modification.

Monacolin K (MK), also known as lovastatin, is a polyketide compound with the ability to reduce plasma cholesterol levels and many other bio-activities. Red yeast rice (also named Hongqu) rich in MK derived from Monascus fermentation has attracted widespread attention due to its excellent performance in reducing blood lipids. However, industrial Monascus fermentation suffers from the limitations such as low yield of MK, long fermentation period, and susceptibility to contamination. In this study, we firstly blocked the competitive pathway of MK biosynthesis to create polyketide synthase gene pigA (the key gene responsible for the biosynthesis of Monascus azaphilone pigments) deficient strain A1. Then, based on the strategies to increase precursor supply for MK biosynthesis, acetyl-CoA carboxylase gene acc overexpression strains C1 and C2 were constructed with WT and A1 as the parent, respectively. Finally, histone deacetylase gene hos2 overexpression strain H1 was constructed by perturbation of histone acetylation modification. HPLC detection revealed all these four strains significantly increased their abilities to produce MK. After 14 days of solid-state fermentation, the MK yields of strains A1, C1, C2, and H1 reached 2.03 g/100 g, 1.81 g/100 g, 2.45 g/100 g and 2.52 g/100 g, which increased by 28.5 %, 14.7 %, 43.9 % and 36.1 % compared to WT, respectively. RT-qPCR results showed that overexpression of hos2 significantly increased the expression level of almost all genes responsible for MK biosynthesis after 5-day growth. Overall, the abilities of these strains to produce MK has been greatly improved, and MK production period has been shortened to 14 days from 20 days, providing new approaches for efficient production of Hongqu rich in MK.

H2O2-Responsive prodrug-nanosystem based on auto-fluorescent perylenetetracarboxylic diimide hinders the foaming progress in RAW264.7 cells.

Oxidative stress can lead to a variety of diseases, and oxalate bonds can consume excess reactive oxygen species (ROS) in cells. In this study, a H2O2-responsive prodrug-nanosystem was synthesized using oxalate-bond-connecting water-soluble drugs (Tre) with a fluorescent indicator (PBI). The maximum fluorescence emission wavelength of PBI-Tre was at 548 nm, and the changes of nanoparticles could be directly observed in the cells. PBI-Tre was coated with hyaluronic acid (HA) to improve their intracellular uptake and ability to target macrophages. The particle size of HA-PBI-Tre was 200-300 nm, and the zeta potential was -36.9 mV. The results showed that the nano-drug loading system could easily decrease the ROS level, inhibit the production of inflammatory factors, remove the accumulation of lipids in foam cells. These nanoparticles could hinder foaming progress in the RAW264. 7 cell line.

A novel electrochemical biosensor for lung cancer-related gene detection based on copper ferrite-enhanced photoinitiated chain-growth amplification.

We reported an electrochemical biosensor via CuFe2O4-enhanced photoinitiated chain-growth polymerization for ultrasensitive detection of lung cancer-related gene. In this work, photoinitiated atom transfer radical polymerization (ATRP) was applied to amplify the electrochemical signal corresponding to lung cancer-related gene, and polymerization was triggered off under the illumination of blue light which was involved in copper-mediated reductive quenching cycle. At the same time, CuFe2O4-H2O2 system was also activated to enhance polymerization based on the photocatalysis of CuFe2O4, which was based on the reaction between •OH and methacrylic monomers to generate carbon-based radicals. Numerous ferrocene-based polymer was graft onto electrode surface through this amplification stages. The limit of detection was low to 1.98 aM (in 10 µL, ∼11.9 molecules) (R2 = 0.998) with a wide linear range from 0.1 fM to 10 pM. This strategy made a good trade-off between cost-effectiveness and sensitivity, and it also presented a high selectivity and anti-interference. In addition, the operation was greatly simplified and detection time was also shortened, which endowed this electrochemical DNA biosensor great application potential.

Structure and dynamics of warm dense aluminum: a molecular dynamics study with density functional theory and deep potential.

We perform a systematic study on the structure and dynamics of warm dense aluminum (Al) at temperatures ranging from 0.5 to 5.0 eV with molecular dynamics utilizing both density functional theory (DFT) and the deep potential (DP) method. On one hand, unlike the Thomas-Fermi kinetic energy density functional (KEDF), we find that the orbital-free DFT method with the Wang-Teter non-local KEDF yields properties of warm dense Al that agree well with the Kohn-Sham DFT method, enabling accurate orbital-free DFT simulations of warm dense Al at relatively low temperatures. On the other hand, the DP method constructs a deep neural network that has a high accuracy in reproducing short- and long-ranged properties of warm dense Al when compared to the DFT methods. The DP method is orders of magnitudes faster than DFT and is well-suited for simulating large systems and long trajectories to yield accurate properties of warm dense Al. Our results suggest that the combination of DFT methods and the DP model is a powerful tool for accurately and efficiently simulating warm dense matter.

CuBr2/EDTA-mediated ATRP for ultrasensitive fluorescence detection of lung cancer DNA.

In this paper, we reported a system for the ultrasensitive fluorescence detection of cytokeratin fragment antigen 21-1 DNA (CYFRA21-1 DNA) for the early diagnosis of lung cancer. The approach used electron transfer atom transfer radical polymerization (ARGET-ATRP) with ethylenediaminetetraacetic acid (EDTA) as the metal ligand. Firstly, thiolated peptide nucleic acid (PNA) was linked to aminated magnetic beads solutions (MBs) by a cross-linking agent and then hybridized with CYFRA21-1 DNA (tDNA). Subsequently, Zr4+ was introduced into the MBs by conjugating with the phosphate group of tDNA, and the initiator of ARGET-ATRP was introduced into via phosphate-Zr4+-carboxylate chemistry. Next, Cu(II)Br/EDTA was reduced to Cu(I)/EDTA by ascorbic acid (AA) to trigger ARGET-ATRP and then a large amount of fluorescein-o-acrylate (FA) molecules were grafted from the surface of the MBs, which amplified significantly the fluorescent signal. Under optimal conditions, a strong linear relationship of tDNA over the range from 0.1 fM to 1 nM (R2 = 0.9988). The limit of detection was as low as 23.8 aM (~143 molecules). The fluorescence detection based on the ARGET-ATRP strategy yielded excellent sensitivity, selectivity, outstanding anti-interference properties, and cost-effectiveness. These results indicated that this strategy has considerable potential for biological detection and early clinical diagnosis.

Dual atom transfer radical polymerization for ultrasensitive electrochemical DNA detection.

Atom transfer radical polymerization as a form of controlled/living radical polymerization is particularly attractive. In this work, dual atom transfer radical polymerization (ATRP) is reported for ultrasensitive DNA detection. Firstly, a peptide nucleic acid (PNA) modified with a thiol group was self-assembled on an electrode surface to capture target DNA (TDNA). The initiator of the first ATRP (ATRP-1), α-bromoisobutyric acid (BIBA), was linked to forming PNA/DNA heteroduplexes via coordination of Zr4+. The polymer chain formed by the monomer of ATRP-1 (2-(2-bromoisobutyryloxy) ethyl methacrylate, BIEM) was also one of initiators of the second ATRP (eATRP-2). The other initiator of eATRP-2 was additional BIBA. ATRP-1 involves activator regeneration by electron transfer (ARGET) ATRP, regulated via excess reducing agent. eATRP-2 is electrochemically mediated ATRP which can control the polymerization via an appropriate applied potential. Compared with one ATRP, more monomers of eATRP-2 modified with ferrocene are attached to electrode surface. Under optimal conditions, this dual ATRP strategy provides a low limit of detection (25 aM, ~150 molecules) with satisfactory selectivity and stability. Importantly, this strategy presents a useful prospect for the field of biomolecule detection.

F-containing initiatior for ultrasensitive fluorescent detection of lung cancer DNA via atom transfer radical polymerization.

An ultrasensitive fluorescence method for early diagnosis of lung cancer via Nafion-initiated atom transfer radical polymerization (ATRP) is reported, in this paper. In the proposed method, thiolated peptide nucleic acid (PNA) is modified to amino magnetic beads (MBs) via a cross-linking agent to specifically capture target DNA (tDNA), and the initiator (Nafion) of ATRP is attached to PNA/DNA heteroduplexes based on the phosphate groups of the tDNA and sulfonate groups of Nafion via phosphate-Zr4+-sulfonate chemistry. Nafion as a macroinitiator of ATRP possesses multiple C-F active sites to initiate polymerization, and numerous polymeric chains that significantly amplify the fluorescent signal are formed. Under optimal conditions, a good linear relationship is obtained in the range of 0.1 nM-0.1 fM with correlation coefficients of 0.9975, and the detection limit is as low as 35.5 aM (∼214 molecules). The proposed strategy has several advantages of simplicity, cost-effectiveness, selectivity and sensitivity. More importantly, the anti-interference results demonstrate that the proposed Nafion-initiated ATRP strategy has great potential in bioanalytical applications.

Dual Signal Amplification by eATRP and DNA-Templated Silver Nanoparticles for Ultrasensitive Electrochemical Detection of Nucleic Acids.

Herein, an ultrasensitive and novel platform for DNA detection is reported, which combines DNA-templated silver nanoparticles (AgNPs) with electrochemical atom transfer radical polymerization signal amplification. Peptide nucleic acid (PNA) functionalized with thiol was modified to the Au electrode surface as a probe to specifically capture target DNA (T-DNA). After Zr4+ binds to phosphate on DNA, the initiator [α-bromophenylacetic acid (BPAA)] of ATRP is attached to PNA/DNA heteroduplexes based on the phosphate groups of T-DNA and carboxylate groups of BPAA via zirconium-phosphate-carboxylate chemistries. A large number of glyco-syloxyethyl methacrylates (GEMA) were captured on the formed PNA/DNA duplex via ATRP. Afterwards, the polysaccharides were oxidized to polymerized aldehydes with sodium periodate (NaIO4). In addition, AgNPs were deposited on the electrode surface by silver mirror reaction. The results indicate that the amount of AgNPs proportional to the T-DNA was quantified through differential pulse voltammetry. Furthermore, it proves that the modified electrode has good performance in DNA detection, indicating that the DNA sensor has high selectivity, high sensitivity, and stable repeatability. Under the optimal conditions, a good linear relationship is obtained in the range of 10 aM to 10 pM with the correlation coefficient of 0.992, and the detection limit is calculated to be as low as 4.725 aM. In addition, the sensor is successfully used to detect DNA in actual serum samples with satisfactory results, which indicates huge promise for detecting gene biomarkers and clinical analysis.

Ultrasensitive DNA biosensor based on electrochemical atom transfer radical polymerization.

Here we report a highly selective and ultrasensitive DNA biosensor based on electrochemical atom transfer radical polymerization (ATRP) signal amplification and "Click Chemistry". The DNA biosensor was prepared by immobilizing thiol and azide modified hairpin DNAs on gold electrode surface. In the presence of target DNAs (T-DNA), hairpin probes hybridized with T-DNAs to form a duplex DNA, and the ring of hairpin DNA was opened to make azide groups accessible at 3' ends. "Click reactions" proceeded between the azide and propargyl-2-bromoisobutyrate (PBIB) to initiate the ATRP reaction which brought a large number of ferrocenylmethyl methacrylate (FMMA) on the electrode surface. The amount of FMMA was proportional to the concentration of T-DNA and quantified by square wave voltammetry. Combining ATRP signal amplification with "Click Chemistry", the optimized DNA biosensor was capable of detecting 0.2 aM. T-DNA. The preliminary application of the developed DNA biosensor was demonstrated by detecting target DNA in spiked serum samples. The developed DNA biosensor shows great promise for the detection of gene biomarkers.