Biomolecule-Driven Two-Factor Authentication Strategy for Access Control of Molecular Devices.

The rise of DNA nanotechnology is promoting the development of molecular security devices and marking an essential change in information security technology, to one that can resist the threats resulting from the increase in computing power, brute force attempts, and quantum computing. However, developing a secure and reliable access control strategy to guarantee the confidentiality of molecular security devices is still a challenge. Here, a biomolecule-driven two-factor authentication strategy for access control of molecular devices is developed. Importantly, the two-factor is realized by applying the specificity and nicking properties of the nicking enzyme and the programmable design of the DNA sequence, endowing it with the characteristic of a one-time password. To demonstrate the feasibility of this strategy, an access control module is designed and integrated to further construct a role-based molecular access control device. By constructing a command library composed of three commands (Ca, Cb, Ca and Cb), the authorized access of three roles in the molecular device is realized, in which the command Ca corresponds to the authorization of role A, Cb corresponds to the authorization of role B, and Ca and Cb corresponds to the authorization of role C. In this way, when users access the device, they not only need the correct factor but also need to apply for role authorization in advance to obtain secret information. This strategy provides a highly robust method for the research on access control of molecular devices and lays the foundation for research on the next generation of information security.

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universal NicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universal NicE-seq on FFPE tissue section sample.

NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells.

A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library preparation. This technology allows cellular level chromatin accessibility quantification and genomic analysis of the epigenetic information in the chromatin, particularly accessible promoter, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding.

Types and Applications of Nicking Enzyme-Combined Isothermal Amplification.

Due to the sudden outbreak of COVID-19 at the end of 2019, rapid detection has become an urgent need for community clinics and hospitals. The rapid development of isothermal amplification detection technology for nucleic acids in the field of molecular diagnostic point-of-care testing (POCT) has gained a great deal of attention in recent years. Thanks to intensive research on nicking enzymes, nicking enzyme-combined isothermal amplification has become a promising platform for rapid detection. This is a novel technique that uses nicking enzymes to improve ordinary isothermal amplification. It has garnered significant interest as it overcomes the complexity of traditional molecular diagnostics and is not subject to temperature limitations, relying on cleavage enzymes to efficiently amplify targets in a very short time to provide a high level of amplification efficiency. In recent years, several types of nicking enzyme-combined isothermal amplification have been developed and they have shown great potential in molecular diagnosis, immunodiagnosis, biochemical identification, and other fields. However, this kind of amplification has some disadvantages. In this review, the principles, advantages and disadvantages, and applications of several nicking enzyme-combined isothermal amplification techniques are reviewed and the prospects for the development of these techniques are also considered.

Assembly of Dynamic Gated and Cascaded Transient DNAzyme Networks.

The dynamic transient formation and depletion of G-quadruplexes regulate gene replication and transcription. This process was found to be related to various diseases such as cancer and premature aging. We report on the engineering of nucleic acid modules revealing dynamic, transient assembly and disassembly of G-quadruplex structures and G-quadruplex-based DNAzymes, gated transient processes, and cascaded dynamic transient reactions that involve G-quadruplex and DNAzyme structures. The dynamic transient processes are driven by functional DNA reaction modules activated by a fuel strand and guided toward dissipative operation by a nicking enzyme (Nt.BbvCI). The dynamic networks were further characterized by computational simulation of the experiments using kinetic models, allowing us to predict the dynamic performance of the networks under different auxiliary conditions applied to the systems. The systems reported herein could provide functional DNA machineries for the spatiotemporal control of G-quadruplex structures perturbing gene expression and thus provide a therapeutic means for related emergent diseases.

Gated Transient Dissipative Dimerization of DNA Tetrahedra Nanostructures for Programmed DNAzymes Catalysis.

Transient dissipative dimerization and transient gated dimerization of DNA tetrahedra nanostructures are introduced as functional modules to emulate transient and gated protein-protein interactions and emergent protein-protein guided transient catalytic functions, operating in nature. Four tetrahedra are engineered to yield functional modules that, in the presence of pre-engineered auxiliary nucleic acids and the nicking enzyme Nt.BbvCI, lead to the fueled transient dimerization of two pairs of tetrahedra. The dynamic transient formation and depletion of DNA tetrahedra are followed by transient FRET signals generated by fluorophore-labeled tetrahedra. The integration of two inhibitors within the mixture of the four tetrahedra and two auxiliary modules, fueling the transient dimerization, results in selective inhibitor-guided gated transient dimerization of two different DNA tetrahedra dimers. Kinetic models for the dynamic transient dimerization and gated transient dimerization of the DNA tetrahedra are formulated and computationally simulated. The derived rate-constants allow the prediction and subsequent experimental validation of the performance of the systems under different auxiliary conditions. In addition, by appropriate modification of the four tetrahedra structures, the triggered gated emergence of selective transient catalytic functions driven by the two pairs of DNA tetrahedra dimers is demonstrated.

Rapid, Visual, and Sequence-Specific Detection of Salmonella in Egg Liquid with vis-NEAA, a CRISPR/Cas12 Empowered New Strategy.

Salmonella is one of the main pathogenic factors that cause foodborne diseases. Rapid and accurate detection of Salmonella in food is of great importance to ensure food safety. Nicking enzyme-assisted amplification (NEAA) is one of the promising isothermal amplification methods finishing the in vitro amplification in ∼10 min; however, it suffers from nonspecific amplification a lot (∼70% products are noises). In this paper, we introduced CRISPR/Cas12a to specifically recognize the NEAA amplicons and transduce the signals into turned-on fluorescent visual readouts (vis-NEAA). Impressively, with this method, the high efficiency of NEAA has been taken great advantage and the nonspecific products were successfully bypassed at the same time. In comparison to NEAA-gel electrophoresis, vis-NEAA showed complete fidelity toward the presence of specific products, while for real-time PCR, it possesses equivalent sensitivity and specificity but saves ∼80% of the time. A level of 80 CFU/mL Salmonella in spiked eggs can be detected on-site in ∼20 min.

Clinical and operational impact of rapid point-of-care SARS-CoV-2 detection in an emergency department.

STUDY OBJECTIVE: Rapid point-of-care (POC) SARS-CoV-2 detection with Abbott ID NOW™ COVID-19 test has been implemented in our Emergency Department (ED) for several months. We aimed to evaluate the operational impact and potential benefits of this innovative clinical pathway. METHODS: We conducted a prospective, descriptive, interventional, non-randomized study, before-after trial with the comparison of patient cohorts from two consecutive periods of seven weeks (observational pre-POC period vs interventional POC period). RESULTS: In 2020, throughout weeks 37 to 50, 3333 patients were assessed for eligibility and among them 331 (9.9%) were positive for SARS-CoV-2 infections. Among the included patients, 136 (9.2%) were positive for SARS-CoV-2 infection in the pre-POC period and 195 (10.5%) in the POC period. Among positive patients for SARS-CoV-2 related infection in-hospital mortality rate was similar between the two groups but the hospitalization rate was higher in the POC group (81.6% vs. 65.4%; p < 0.001). More patients in the POC period were able to leave the ED within 6 h. We examined rates of antibiotic, anticoagulant, and corticosteroid prescriptions among patients tested for SARS-CoV-2 in the ED. Only the rate of prescribed anticoagulants was found to be higher in the POC period (40% vs. 24.2%; p < 0.003). CONCLUSION: We demonstrated that COVID-19 point-of-care testing speeds up clinical decision-making, improving use of recommended treatments for COVID-19, such as anticoagulants. Moreover, it improves the boarding time and significantly shortened the length of stay in the ED for patients requiring outpatient care.

Engineering entropy-driven based multiple signal amplification strategy for visualized assay of miRNA by naked eye.

MicroRNAs (miRNAs) are currently recognized as novel biomarkers for cancer early diagnosis, therapy selection, and progression monitoring. Herein, we developed an ultrasensitive and label-free homogeneous colorimetric strategy for miRNA detection based on engineering entropy-driven amplification (EDA) coupled with nicking enzyme-assisted AuNP aggregation. In our design, the target miRNA could specifically trigger the EDA recycling process. One of the EDA products could open the hairpin probe and form a dual strand containing a nicking endonuclease (Nb.BbvCl) cleavage region. After adding nicking endonuclease in the sensing solution, the product DNA fragments could act as two linkers, inducing the aggregation of ssDNA-modified AuNPs. Simultaneously, the liberating complementary strands continued to cyclic hybridization with the hairpin probe. This multiple signal amplification colorimetric strategy showed a wide linear range from 10 fM to 100 pM with a much lower detection limit of 3.13 fM for miRNA let-7a, which also performed well in a complex sample matrix. Most importantly, the naked eye could clearly distinguish the 10 fM color change caused by let-7a to be measured. Moreover, this approach could easily extend to multiple miRNAs with target-specific sequence substitutions. Therefore, this ultrasensitive visual strategy for miRNA demonstrated attractive potentials for promising applications in clinical diagnosis.

Integrated nicking enzyme-powered numerous-legged DNA walker prepared by rolling circle amplification for fluorescence detection of microRNA.

MicroRNAs (miRNAs) have been accepted as promising non-invasive biomarkers for cancer early diagnosis. Developing amplified sensing strategies for detecting ultralow concentration of miRNAs in clinical samples still requires much effort. Herein, an integrated fluorescence biosensor using nicking enzyme-powered numerous-feet DNA walking machine was developed for ultrasensitive detection of miRNA. A long numerous-feet walker produced by target-triggered rolling circle amplification autonomously moves along the defined DNA tracks on gold nanorods (AuNRs) with the help of nicking enzyme, leading to the recovery of fluorescence. This results in an amplified fluorescence signal, typically measured at 518 nm emission wavelength. Benefiting from the long walker that dramatically improves movement range, the homogenous and one-step strategy realizes ultrahigh sensitivity with a limit of detection of 0.8 fM. Furthermore, this walking machine has been successfully used to quantification of miRNA in clinical serum samples. The consistency of the gained results between of the developed strategy and reverse transcription quantitative polymerase chain reaction (RT-qPCR) shows that the sensing method has great promise for tumor diagnostics based on nucleic acid. Schematic representation of the fluorescent biosensing strategy, numerous-legged DNA walker prepared by rolling circle amplification on gold nanorods (AuNRs) for microRNA analysis, which can be applied in real samples with good results.

Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain.

Strand-specific DNA nicking endonucleases (NEases) typically nick 3-7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5' GGATCC-N(4-6)-AC↑CGR 3' (R, A, or G) or to nick both sides of BamHI site at the composite site 5' CCG↓GT-N5-GGATCC-N5-AC↑CGG 3' (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn2+ or Ni2+, and it has low activity in Mg2+ buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications.

Aptamer biorecognition-triggered hairpin switch and nicking enzyme assisted signal amplification for ultrasensitive colorimetric bioassay of kanamycin in milk.

A colorimetric aptasensing strategy for detection of kanamycin was designed based on aptamer biorecognition and signal amplification assisted by nicking enzyme. The aptamer of kanamycin was designed to be contained in the metastable state hairpin DNA. The target DNA as recycling DNA was located in the loop of hairpin DNA. The presence of kanamycin stimulates the continuous actions, including specific recognition of the aptamer to kanamycin, the hybridization between target DNA and signal probe, the cleavage function of nicking enzyme. The actions induced accumulation of numerous free short sequences modified by platinum nanoparticles (PtNPs), which can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 to produce a colorimetric response. The aptasensor exhibited good selectivity and sensitivity for kanamycin in milk with a detection limit as low as 0.2 pg·mL-1. In addition, the proposed assay is potentially to be extended for other antibiotics detection in foods by adapting the corresponding aptamer sequence.

Toehold-mediated strand displacement reaction formation of three-way junction DNA structure combined with nicking enzyme signal amplification for highly sensitive colorimetric detection of Salmonella Typhimurium.

The detection of Salmonella Typhimurium (S.typhimurium) is of great importance in food safety field. Colorimetric strategy is particularly appealing for S. typhimurium identification because of its user-friendliness and instrument-free. However, the existing colorimetric strategies still meet the challenges of low sensitivity, tedious nucleic acid extraction and expensive labeling processes. Herein, a high sensitivity and label-free colorimetric sensing strategy for S. typhimurium detection without nucleic acid extraction is constructed. Specifically, the proposed strategy is based on three-way junction (3WJ) DNA branched structure combined with nicking enzyme signal amplification (NESA). In the presence of target, cascaded signal amplification is initiated through a series of toehold-mediated strand displacement reactions (TSDRs) to recycle the trigger DNA causing formation of the numerous 3WJ DNA branched structures (3WJ-TSDRs). Then, the branches of 3WJ-TSDRs are fully utilized to hybridize with the DNAzyme signal probes to initiate NESA in the presence of Nt. BbvCI, which making every branch has a function of signal amplification. Finally, DNAzyme signal probes (green) were completely split into two fragments (colorless). The application of NESA in the branches of 3WJ-TSDRs offers a highly sensitive detection of S. typhimurium with a low limit of detection of 42 CFU mL-1. Besides, the colorimetric sensing strategy also shows strong anti-interference. The capability of the colorimetric sensing strategy in spiked samples was also investigated, showing a more intuitive results and fast detection in compare with the traditional plate counting method. With these characteristics, the proposed sensing strategy based on 3WJ-TSDRs and NESA is a promising tool for new point-of-care (POC) applications in food safety.

Construction of a cDNA expression library in a binary vector using a nicking enzyme.

Ligation-independent cloning (LIC), such as Gibson Assembly, tends to produce clones without an insert, depending on the sequences present at the ends of linearized vectors. We used a nicking enzyme-mediated LIC (NE-LIC) method to construct a cDNA library in a binary vector pER8. Prior to constructing the cDNA library, pilot experiments were carried out, in which the GUS coding sequence was cloned into pER8 using NE-LIC. Approximately 12% of input vector DNAs were converted to plasmids carrying a GUS insert, and no plasmids without an insert were detected, indicating that this strategy is highly effective for cloning with the binary vector pER8. Therefore, NE-LIC was adopted to construct a cDNA library in pER8, by using cDNA that was PCR-amplified from a library constructed in another vector. As a result, a cDNA library in pER8 was successfully constructed. During library construction, it is important to exclude plasmids without an insert, since contamination from plasmids without inserts decreases the efficiency of screening. Therefore, NE-LIC is useful for the construction of cDNA libraries.

CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase.

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10-5 to 2.5 × 10-2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10-6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening.

A molecular device: A DNA molecular lock driven by the nicking enzymes.

As people are placing more and more importance on information security, how to realize the protection of information has become a hotspot of current research. As a security device, DNA molecular locks have great potential to realize information protection at the molecular level. However, building a highly secure molecular lock is still a serious challenge. Therefore, taking advantage of the DNA strand displacement and enzyme control technology, we constructed a molecular lock with a self-destructive mechanism. This molecular lock is mainly composed of logic circuits and takes nicking enzymes as inputs. To build this molecular lock, we first constructed a series of cascade circuits, including a YES-YES cascade circuit and a YES-AND cascade circuit. Then, an Inhibit logic gate was constructed to explore the inhibitory properties between different combinations of two nicking enzymes. Finally, using the characteristics of mutual inhibition between several enzymes, a DNA molecular lock driven by three nicking enzymes was constructed. In this molecular device, only the correct sequence of nicking enzymes can be input to ensure the normal operation of the molecular lock. Once the wrong password is entered, the device will be destroyed and cannot be recovered, which effectively prevents intruders from cracking the lock through exhaustive methods. The molecular lock has the function of simulating an electronic keyboard, which can realize the protection of information at the molecular level, and provides a new implementation method for building more advanced and complex molecular devices.

Visualization and Sequencing of Accessible Chromatin Reveals Cell Cycle and Post-HDAC inhibitor Treatment Dynamics.

Chromatin accessibility is a predictor of gene expression, cell division, and cell type specificity. NicE-viewSeq (Nicking Enzyme-assisted viewing and Sequencing) allows accessible chromatin visualization and sequencing with overall lower mitochondrial DNA and duplicated sequences interference relative to ATAC-see. Using NicE-viewSeq, we interrogated the accessibility of chromatin in a cell cycle (G1, S, and G2/M)-specific manner using mammalian cells. Despite DNA replication and subsequent condensation of chromatin to chromosomes, chromatin accessibility remained generally preserved with minimal subtle alterations. Genome-wide alteration of chromatin accessibility within TSS and enhancer elements gradually decreased as cells progressed from G1 to G2M, with distinct differential accessibility near consensus transcription factors sites. Inhibition of histone deacetylases promoted accessible chromatin within gene bodies, correlating with apoptotic gene expression. In addition, reduced chromatin accessibility for the MYC oncogene pathway correlated with downregulation of pertinent genes. Surprisingly, repetitive RNA loci expression remained unaltered following histone acetylation-mediated increased accessibility. Therefore, we suggest that subtle changes in chromatin accessibility are a prerequisite during the cell cycle and histone deacetylase inhibitor-mediated therapeutics.

Target-triggered configuration change of DNA tetrahedron for SERS assay of microRNA 122.

A surface-enhanced Raman scattering (SERS) method is proposed for the assay of microRNA 122 based on configuration change of DNA tetrahedron. Firstly, a DNA tetrahedron was self-assembled with one vertex labeled with toluidine blue (TB). Then, it was immobilized on the porous Ni/SiO2@PEI@Au as a SERS platform, which was characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). At this time, the DNA tetrahedron was contracted; so, the TB is close to AuNPs and the Raman signal is high. When target microRNA 122 existed, with the nicking enzyme amplification strategy, a great deal of DNA signal chains (S5) was obtained, which can extend the contracted DNA tetrahedron and change it into a three-dimensional DNA tetrahedron. In this case, the TB was far from AuNPs, resulting in a lower Raman signal. Due to the configuration change of DNA tetrahedron, the Raman signal at 1624 cm-1 (with the excitation wavelength of 633 nm) has a linear relationship with the logarithm concentration of microRNA 122. This SERS assay has high sensitivity for microRNA 122 with a determination range from 0.01 aM to 10 fM and a detection limit of 0.009 aM. The recoveries from spiked samples were in the range 95 to 109%. This SERS strategy is designed based on the target-triggered configuration change of DNA tetrahedron, which can give new insight for DNA structures in bioanalysis. Graphical abstract A sensitive surface-enhanced Raman scattering (SERS) biosensor was developed to detect microRNA 122 using the configuration change of DNA tetrahedron to indirectly control the position of TB and hot spot.

An All-in-One Aptasensor Integrating Enzyme Powered Three-Dimensional DNA Machine for Antibiotic Detection.

In this work, we have developed an all-in-one aptasensor based on an enzyme-driven three-dimensional DNA walker for antibiotic detection. To overcome the drawback of time-consuming methods, high-density substrate strands were anchored on the walking interface that accelerated the signal amplification efficiency. Such an all-in-one design integrated the functionality of target recognition, signal amplification, as well as signal output into a single probe. Upon addition of kanamycin, the activated walking strand moved along the track by the stepwise cleavage of a nicking enzyme, which resulted in the enhancement of the fluorescence intensity of the solution. Under the optimized conditions, the detection process was accomplished in 40 min with a low detection limit of 1.23 pM. This aptasensor was also applied in spiked milk samples with satisfactory recoveries of 97.76% to 105.33%, demonstrating an excellent stability and accuracy. Therefore, this all-in-one aptasensor shows great potential for applications in food safety.

A rapid, adaptative DNA biosensor based on molecular beacon-concatenated dual signal amplification strategies for ultrasensitive detection of p53 gene and cancer cells.

The cancer diagnosis with single level of biomarkers suffers from limitation of insufficient accuracy. Hence, developing sensitive, rapid and adaptative analytical strategies for double-level biomarkers are essential for improving the accuracy of clinical cancer diagnosis at early stage. Herein, a DNA biosensor was established based on the catalytic hairpin assembly-mediated Y-junction nicking enzyme assisted signal amplification (CHA-YNEASA) circuits, where the two circuits were concatenated by molecular beacon (MB). In absence of target, both the CHA and YNEASA circuits were effectively hindered because of MB's outstanding ability to control signal background. In presence of target, the initiated CHA circuits made enzyme recognition sequences in close proximity to the assisted sequences to open MB, leading to further trigger the YNEASA circuits. Due to the unique design of dual signal amplification strategies, CHA-YNEASA circuits significantly shorten the reaction time, and improve signal-to-background ratio as well as facilitate the analysis process. It was demonstrated that a high sensitivity with limit of detection (LOD) of 0.9 pM for p53 gene detection was obtained just within 23 min by the proposed DNA biosensor. Moreover, mismatched p53 gene at nucleic acid level was effectively discriminated and strong anti-interference capability was achieved. Noticeably, the DNA biosensor was adaptative for designing a cytosensor at cell level using hairpin DNA, containing MUC1 aptamer and initiation strand of CHA-YNEASA circuits, as switch based on modularity principle. The cytosensor is able to measure MUC1 positive breast cancer cells (MCF-7) with the LOD as low as 100 cells/mL. Excellent specificity for MUC1 negative cells, and good anti-interference capability in 10% fetal bovine serum (FBS) were observed by the cytosensor. Therefore, the proposed DNA biosensor is a sensitive, rapid, adaptative platform for detection of double-level biomarkers, offering novel strategy applied for clinical cancer diagnosis.