Single-base Mismatch Research Articles

Single-Molecule Detection of Serum MicroRNAs for Medulloblastoma with Biphasic Sandwich Hybridization-Assisted Plasmonic Resonant Scattering Imaging.

MicroRNA (miRNA) dysregulation is closely related to the occurrence and progression of medulloblastoma (MB). However, the full potential of serum circulating miRNAs in MB diagnosis is restricted by their ultralow abundance in peripheral blood due to blood-brain barrier. Here, we report the direct preamplification-free detection of aberrant expression of oncogenic miRNAs in serum from MB patients by proposing a simple yet robust single-molecule assay that combines biphasic sandwich hybridization in nucleic acids and the dark-field single-particle plasmonic imaging (B2S2PI). In this strategy, signal DNA was prehybridized with target miRNA in homogeneous solution to form sDNA-RNA complexes. Then the captured DNA strands with rationally adjusted surface densities could efficiently capture the sDNA-RNA complexes to generate a well-separated DNA-RNA sandwich structure. The combination of homogeneous and heterogeneous reactions enabled interface-mediated hybridization reactions to maintain molecular stability with fewer bases, making it suitable for the direct amplification-free assays of short miRNA targets. Labeling the DNA-RNA hybrids with plasmatic gold nanotags allowed nondestructive recognition and imaging of individual miRNA targets under mild conditions with high signal-to-noise ratio. By digitally counting and analyzing the bright plasmonic resonant scattering spots, B2S2PI enabled both the measurement of a low femtomolar concentration of circulating miRNA-21 in 5 μL sample volume within a turnaround of 2 h and the discrimination of single base mismatches. Moreover, B2S2PI was universal for detecting miRNAs with different sequences and secondary structures. Further analysis of clinical serum samples revealed that B2S2PI was capable of accurately distinguishing MB patients from noncancer controls with an area under the curve (AUC) of 0.99, which was superior to that of qRT-PCR. B2S2PI holds promise as a novel alternative means for single-molecule miRNA assay and sheds light on the circulating nucleic acid-based liquid biopsy of intracranial malignant tumors.

Binding-driven forward tearing protospacer activated CRISPR-Cas12a system and applications for microRNA detection

CRISPR-Cas12a system, characterized by its precise sequence recognition and cleavage activity, has emerged as a powerful and programmable tool for molecular diagnostics. However, current CRISPR-Cas12a-based nucleic acid detection methods, particularly microRNA (miRNA) detection, necessitate additional bio-engineering strategies to exert control over Cas12a activity. Herein, we propose an engineered target-responsive hairpin DNA activator (TRHDA) to mediate forward tearing protospacer activated CRISPR-Cas12a system, which enables direct miRNA detection with high specificity and sensitivity. Target miRNA specifically binding to hairpin DNA can drive forward tearing protospacer in the stem sequence of hairpin structure, facilitating the complementarity between crRNA spacer and protospacer to activate Cas12a. Upon the hairpin DNA as input-responsive activator of Cas12a, a universal biosensing method enables the multiple miRNAs (miR-21, let-7a, miR-30a) detection and also has exceptional capability in identifying single-base mismatches and distinguishing homologous let-7/miR-30 family members. Besides, TRHDA-mediated Cas12a-powered biosensing has realized the evaluation of miR-21 expression levels in diverse cellular contexts by intracellular imaging. Considering the easy programmability of hairpin DNA in responsive region, this strategy could expand for the other target molecules detection (e.g., proteins, micromolecules, peptides, exosomes), which offers significant implications for biomarkers diagnostics utilizing the CRISPR-Cas12a system toolbox.Graphical abstract

Single nucleotide polymorphism discrimination and genotyping based on cascade strand displacement reaction mediated label-free Cas12a system

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy.

Characterization of argonaute nucleases from mesophilic bacteria Pseudobutyrivibrio ruminis

Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.Graphical

A Triple-Mismatch Differentiating assay exploiting activation and trans cleavage of CRISPR-Cas12a for mutation detection with ultra specificity and sensitivity

Liquid biopsy technology is non-invasive and convenient, and is currently an emerging technology for cancer screening. Among them, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12a (Cas12a) based nucleic acid detection technology has the advantages of high sensitivity, rapidity, and easy operation. However, CRISPR-Cas12a does not discriminate single-base mismatches of targets well enough to meet the needs of clinical detection. Herein, we developed the Triple-Mismatch Differentiating (TMD) assay. This assay amplified the small thermodynamic difference in mismatches at one site at the level of CRISPR-Cas12a activation to a significant thermodynamic difference at three sites at both the level of CRISPR-Cas12a activation and trans-cleavage, which greatly improves the ability of CRISPR-Cas12a to discriminate between base mismatches. Our manipulation greatly improved the specificity of the CRISPR-Cas12a system while maintaining its inherent sensitivity and simplicity, increasing the detection limit to 0.0001%. When testing samples from pancreatic cancer patients, our results were highly consistent with NGS sequencing results. We believe that the TMD assay will provide a new technology for early cancer detection and will be widely used in the clinical practice.

Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors.

An enzyme-free and label-free multiplex detection of miRNAs by entropy-driven circuit coupled with capillary electrophoresis

MicroRNAs (miRNAs) are currently recognized as important biomarkers for the early diagnosis and prognostic treatment of cancer. Herein, we developed a simple and label-free method for the multiplex detection of miRNAs, based on entropy-driven circuit (EDC) amplification and non-gel sieving capillary electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform. In this system, three different lengths of fuel chains were designed to catalyze three EDC, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. In the presence of target miRNA, the EDC cycle amplification reaction was triggered, generating numerous stable double-strands products (F-DNA/L-DNA). Since the three miRNAs correspond to three different lengths of F-DNA/L-DNA, they can be easily isolated and detected by NGCE. This strategy has good sensitivity, with detection limits of 68 amol, 292.2 amol, and 394 amol for miRNA-21, miRNA-155, and miRNA-10b, respectively. Additionally, this method has good specificity and can effectively distinguish single-base mismatches of miRNA. The recoveries of the three miRNAs in deproteinized healthy human serum ranged from 91.28 % to 108.4 %, with a relative standard deviation (RSD) of less than 7.9 %. This method was further applied to detect cellular miRNAs in human breast cancer (MCF-7) cell extracts, revealing an up-regulation of miRNA-21, miRNA-155, and miRNA-10b in MCF-7 cells. The successful spiked recovery in human serum and RNA extraction from MCF-7 cells underscores the practicality of this method. Therefore, this strategy has broad application prospects in biomedical research.

Ultra-sensitive DNA detection with single-base mismatch resolution using a tapered thin-diameter photonic crystal fiber interferometer

A tapered thin-diameter photonic crystal fiber interferometer (TTD-PCFI) biosensor is proposed and demonstrated for DNA hybridization detection with an ultra-low limit of detection (LoD) and high specificity. The TTD-PCFI is fabricated by arc-discharged melting and adiabatic tapering, with a lower loss and stronger evanescent optical field. The fabricated TTD-PCFI with a diameter of 8.63 µm achieves a high refractive index sensitivity of 3842.5 nm/RIU. DNA hybridization detection of concentration ranging from 1 fM to 1 pM is achieved, with a sensitivity of 632 pm/lg(fM). The measured LoD is as low as 410 aM. Single-base mismatch detection of 25-base target single-stranded DNA is also achieved. The proposed label-free DNA biosensor has the advantages of the ultra-low LoD, high specificity, and real-time detection, which has great application prospects in the fields of disease diagnosis, microbial detection, and environmental science.

High-performance electrolyte-gated amorphous InGaZnO field-effect transistor for label-free DNA sensing

Accurate, convenient, label-free, and cost-effective biomolecules detection platforms are currently in high demand. In this study, we showcased the utilization of electrolyte-gated InGaZnO field-effect transistors (IGZO FETs) featuring a large on-off current ratio of over 106 and a low subthreshold slope of 78.5 mV/dec. In the DNA biosensor, the modification of target DNA changed the effective gate voltage of IGZO FETs, enabling an impressive low detection limit of 0.1 pM and a wide linear detection range from 0.1 pM to 1 μM. This label-free detection method also exhibits high selectivity, allowing for the discrimination of single-base mismatch. Furthermore, the reuse of gate electrodes and channel films offers cost-saving benefits and simplifies device fabrication processes. The electrolyte-gated IGZO FET biosensor presented in this study shows great promise for achieving low-cost and highly sensitive detection of various biomolecules.

Pitfalls and challenges of peptide nucleic acid immobilisation on carbon surfaces for sequence-specific capturing of nucleic acid biomarkers

Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA's (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe.

A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

Biosensors for Public Health and Environmental Monitoring: The Case for Sustainable Biosensing.

Climate change is a profound crisis that affects every aspect of life, including public health. Changes in environmental conditions can promote the spread of pathogens and the development of new mutants and strains. Early detection is essential in managing and controlling this spread and improving overall health outcomes. This perspective article introduces basic biosensing concepts and various biosensors, including electrochemical, optical, mass-based, nano biosensors, and single-molecule biosensors, as important sustainability and public health preventive tools. The discussion also includes how the sustainability of a biosensor is crucial to minimizing environmental impacts and ensuring the long-term availability of vital technologies and resources for healthcare, environmental monitoring, and beyond. One promising avenue for pathogen screening could be the electrical detection of biomolecules at the single-molecule level, and some recent developments based on single-molecule bioelectronics using the Scanning Tunneling Microscopy-assisted break junctions (STM-BJ) technique are shown here. Using this technique, biomolecules can be detected with high sensitivity, eliminating the need for amplification and cell culture steps, thereby enhancing speed and efficiency. Furthermore, the STM-BJ technique demonstrates exceptional specificity, accurately detects single-base mismatches, and exhibits a detection limit essentially at the level of individual biomolecules. Finally, a case is made here for sustainable biosensors, how they can help, the paradigm shift needed to achieve them, and some potential applications.

Advancing COVID-19 Detection: High-Performance RNA Biosensing via Electrical Interactions

This research paper investigated the detection of COVID-19 using an Aluminum Interdigitated Electrode (Al-IDE) sensor based on electrical conductivity. The silanization process involved the functionalization step, employing (3-Aminopropyl) triethoxysilane (APTES), while the immobilization process was facilitated by the RNA Probe specific to COVID-19. To verify its specificity in detection, the functionalized biosensor was tested against single-base mismatches, non-complementary sequences, and complementary sequences. The physical characteristics of the Al-IDE biosensor were examined using both low-power microscopy (LPM) and high-power microscopy (HPM). Additionally, the morphological properties of the biosensor were assessed using atomic force microscopy (AFM). To assess its diagnostic potential, the biosensor's sensitivity was evaluated by exposing it to a range of complementary targets, spanning from 1 femtomolar (fM) to 1 micromolar (μM). The current-voltage (I-V) characteristics of the biosensor were meticulously analyzed at each stage of functionalization bare Al-IDE, silanization, immobilization, and hybridization. This I-V characterization was carried out using a picoammeter voltage source (Keithley 2450), Kickstart software, and a probe station. The results confirmed the biosensor's capability to effectively detect COVID-19 targets within the nanoampere concentration range, demonstrating its success in detecting specific COVID-19 targets at the nanoampere level.

ACURAT: Advancing tuberculosis detection through assembled PCR & CRISPR for ultra-sensitive Rifampin-resistant analysis testing

Tuberculosis (TB) remains a pressing global health threat, while the emergence of drug-resistant TB adds complexity to clinical treatment strategies, underscoring the need for rapid and accurate diagnosis. In this study, we present an innovative identification and antibiotic susceptibility testing system, termed Assembled PCR & CRISPR for Ultra-sensitive Rifampin-resistant Analysis Testing (ACURAT). ACURAT employed a nested polymerase chain reaction (PCR) scheme to sensitively detect as few as 10 copies of the TB genome, more importantly, addressing the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR-Cas9) system’s requirement for a protospacer adjacent motif (PAM) sequence by introducing with the designed primers. Recognizing the limitation of fully-matched guide ribonucleic acid (RNA) in distinguishing one-base single nucleotide polymorphisms (SNPs), we designed a micro-mismatch CRISPR-Cas9 system with significant selectivity, featuring a single-base mismatch with wildtype genotypes and at least two base mismatches with drug-resistant genotypes. The systematic optimization of ACURAT parameters revealed that a 19 nt guide RNA length, a 1 bp distance to the PAM sequence, and the use of a high-fidelity Cas9 nuclease significantly enhanced the efficiency. Finally, ACURAT successfully distinguished fifteen drug-resistance-related genotypes in codons 516, 526, 531, and 533 of the polymerase beta subunit gene. ACURAT demonstrated substantial clinical potential for efficient TB diagnosis and exhibited the capability to address other sequence recognition scenarios.

Cascade CRISPR/Cas12a and DSN for the electrochemical biosensing of miR-1246 in BC-derived exosomes

MiR-1246 in breast cancer-derived exosomes was a promising biomarker for early diagnosis of breast cancer(BC). However, the low abundance, high homology and complex background interference make the accurate quantitative detection of miR-1246 facing great challenges. In this study, we developed an electrochemical biosensor based on the subtly combined of CRISPR/Cas12a, double-stranded specific nuclease(DSN) and magnetic nanoparticles(MNPs) for the detection of miR-1246 in BC-derived exosomes. Ascribed to the good synergistic effect of DSN, Cas12a and MNPs, the developed electrochemical biosensor exhibited excellent performance with the linear range from 500 aM to 5 pM, and the detection limit as low down to about 50 aM. The target-specific triggered enzyme-digest activity of DSN and Cas12a system, as well as the powerful separation ability of MNPs ensure the high specificity of developed electrochemical biosensor which can distinguish single base mismatches. In addition, the developed electrochemical biosensor has been successfully applied to detect miR-1246 in blood-derived exosomes and realize distinguishing the BC patients from the healthy individuals. It is expected that the well-designed biosensing platform will open up new avenues for clinical liquid biopsy and early screening of breast cancer, as well as provide deeper insights into clinical oncology treatment.

Development of SPQC sensor based on the specific recognition of TAL-effectors for locus-specific detection of 6-methyladenine in DNA

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance.

Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells.

As an important disease biomarker, the development of sensitive detection strategies for miRNA, especially intracellular miRNA imaging strategies, is helpful for early diagnosis of diseases, pathological research, and drug development. Hybridization chain reaction (HCR) is widely used for miRNA imaging analysis because of its high specificity and lack of biological enzymes. However, the classic HCR reaction exhibits linear amplification with low efficiency, limiting its use for the rapid analysis of trace miRNA in living cells. To address this problem, we proposed a toehold-mediated exponential HCR (TEHCR) to achieve highly sensitive and efficient imaging of miRNA in living cells using β-FeOOH nanoparticles as transfection vectors. The detection limit of TEHCR was as low as 92.7 fM, which was 8.8 × 103 times lower compared to traditional HCR, and it can effectively distinguish single-base mismatch with high specificity. The TEHCR can also effectively distinguish the different expression levels of miRNA in cancer cells and normal cells. Furthermore, TEHCR can be used to construct OR logic gates for dual miRNA analysis without the need for additional probes, demonstrating high flexibility. This method is expected to play an important role in clinical miRNA-related disease diagnosis and drug development as well as to promote the development of logic gates.

Enhanced Sequence-Specific DNA Recognition Using Oligodeoxynucleotide-Benzimidazole Conjugates.

Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.

Cooperative activation of CRISPR-Cas12a based on optimization of nucleic acid hybridization kinetics and thermodynamics: More specific, flexible and controllable

The high efficiency of CRISPR-Cas12a is highly beneficial for rapid and sensitive detection of low-concentration markers. However, its sloppy identification and uncontrollable high activity lead to a significant loss in specificity, flexibility, and expandability. In this paper, we present a cooperative activation strategy that is both simple and powerful, based on the kinetic and thermodynamic mechanisms of nucleic acid hybridization reactions. Using the EGFR T790M and L858R mutant as models, we study base by base in detail and demonstrate that the cooperative activation strategy has strong general properties that can be applied to various sequence systems. Utilizing the sub-stable state of hybridization to effectively improve the specificity of single-base mismatches of activators, and also dramatically improves the design flexibility and utilization efficiency of crRNAs by cooperating with different sequences. In addition, this strategy also creatively uses the strand displacement reaction to terminate the trans-activity at any time, and more importantly, such inhibition is reversible, which undoubtedly greatly improves the controllability of the CRISPR-Cas12a system, and enables it to have a more and more abundant functional expansion space. Overall, our strategy has obvious advantages, which will unleash the potential for expanding and deepening the application of CRISPR technology in molecular biology.

Label-free and portable detection of HIV-DNA by a handheld luminometer

BackgroundThe human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. ResultsIn this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05–10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SignificanceIn-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.