Hairpin Probe Research Articles

Hairpin/DNA Ring Ternary Complex Initiated Rolling Circle Amplification for an Elevated Accuracy and Its Application in Analyzing Let-7a.

Rolling circle amplification (RCA) is an attractive isothermal nucleic acid amplification approach and has been widely applied in constructing a variety of biosensors. However, the inevitable drawbacks of lacking enough selectivity greatly hindered further applications of RCA-based approaches. Here, we develop a novel RCA-based approach by integrating the specific target recognition capability of the hairpin/DNA ring ternary complex and multiple signal amplification and successfully applied it for let-7a detection. In this method, let-7a specifically unfolds the hairpin probe (Hp probe) in the ternary complex to induce target recycle and RCA- and DNAzyme-based signal generation. Based on this, the established approach exhibits a high selectivity to let-7a, and the response of the approach to one base pair mismatched sequences was 24.9%, indicating a significantly improved specificity. Meanwhile, the limit of detection is as low as 342 aM, which can meet the high requirement for a trace amount of miRNA detection. In all, we believe that the established approach can offer a new avenue for miRNA detection and post-tumor care.

Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design.

Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%-0.13%, and those for analytical limit of detection were 0.00%-0.20%. Inter-laboratory reproducibility was excellent (r 2 = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings.

Photocontrollable DNA Walker-Based Molecular Circuit for the Tunable Detection of MicroRNA-21 Using Metal-Organic Frameworks as Label-Free Fluorescence Tags.

Tunable detection of microRNA is crucial to meet the desired demand for sample species with varying concentrations in clinical settings. Herein, we present a DNA walker-based molecular circuit for the detection of miRNA-21 (miR-21) with tunable dynamic ranges and sensitivity levels ranging from fM to pM. The phosphate-activated fluorescence of UiO-66-NH2 metal-organic framework nanoparticles was used as label-free fluorescence tags due to their competitive coordination effect with the Zr atom, which significantly inhibited the ligand-to-metal charge transfer. To achieve a tunable detection performance for miR-21, the ultraviolet sensitive o-nitrobenzyl was induced as a photocleavable linker, which was inserted at various sites between the loop and the stem of the hairpin probe to regulate the DNA strand displacement reaction. The dynamic range can be precisely regulated from 700- to 67,000-fold with tunable limits of detection ranging from 2.5 fM to 36.7 pM. Impressively, a Boolean logic tree and complex molecular circuit were constructed for logic computation and cancer diagnosis in clinical blood samples. This intelligent biosensing method presents a powerful solution for converting complex biosensing systems into actionable healthcare decisions and will facilitate early disease diagnosis.

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

Enzymes-mediated facile biosynthesis of G-quadruplexes for sensitive detection of apurinic/apyrimidinic endonuclease 1 activity

Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential role in regulating physiological functions and is a critical node in tumor progression. However, conventional detection methods for discriminating APE1 activity are hampered by labor-intensive procedures, multiple operations, and limited detection sensitivity. Herein, we have developed a terminal deoxynucleotidyl transferase (TdT)-mediated randomly arrayed G-quadruplexes (G4s) biosynthesis assay (TGBA) for facile and sensitive detection of APE1 activity. In this assay, the recognition DNA hairpin probe (rDHP) with an AP-site in the stem and modification of the phosphate group in 3′-end (3′-P) has been well-designed to be specifically cleaved by APE1 and then expose the new 3′-OH for rDHP template-free elongation by TdT. Following the rational proportion of the G-rich dNTP pool, the amplified and well-formed G4s can be obtained to bind to the G4 fluorescent dye, such as Thioflavin T (ThT). The effective, specific, high-contrast, and rapid fluorescence readout of TGBA has been validated by the in vitro analysis of APE1 activity and the evaluation of APE1 inhibitor, which enables the sensitive detection of APE1 activity with a lower detection limit of 0.001 U/mL and the detection linear range from 0.005 to 1 U/mL. Inspired by these results, we seek to explore the analysis of APE1 activity in different scenarios, including human serum and cell lysates. The unambiguously distinguishing capability of TGBA is proved by both detecting endogenous APE1 activity in cell lysates and exogenous APE1 activity in human sera. Collectively, TGBA will exhibit promising potential in the diagnosis of APE1-related diseases and the evaluation of drug efficacy.

Programmable SARS-CoV-2 mutation detection platform based on ligation-triggered isothermal exponential amplification coupled with self-priming amplification

In this work, we developed a programmable mutation detection platform with a dual amplification strategy for universal detection of SARS-CoV-2 mutations based on ligation-triggered isothermal exponential amplification reaction(L-EXPAR) and phosphorothioate modified self-priming amplification (PS-SPA). In the presence of mutant target (MT) sequence, it complements with hairpin probe (H1) and hairpin probe (H2) and triggered L-EXPAR at lower temperature. A large number of single-strand DNA (ssDNA) is produced in the circle of ligation, nicking, polymerization, and strand displacement. The ssDNA products containing target sequences can feed back and initiate another L-EXPAR. The ligated H1-H2 complex triggers another isothermal process termed PS-PSA at higher temperature. 20 bases of H1-H2 complex on the 5′ end modified with PS can form a short stem-loop hairpin due to the lower Tm of PS modification. The resulting overhanging 3′ end can also fold back as a new primer to initiate the next round of polymerization. In the circle of PS-SPA, the H1-H2 chain can be extended to be a long hairpin concatemer containing a great number of tandem target sequences. The resulting DNA products can be monitored in real-time fluorescent signaling. In the presence of the SARS-CoV-2 wild type (WT) sequence, the ligation between H1 and H2 cannot happen due to the mismatch of the last base on the 3′ end of H1. The proposed assay displayed a good linear range from 1 fM to 1 nM with a detection limit of femtomole level for the detection of SARS-CoV-2 spike gene D614G mutation and Del246-252 + D253N deletion.

Orderly Aggregated Catalytic Hairpin Assembly for Synchronous Ultrasensitive Detecting and High-Efficiency Co-Localization Imaging of Dual-miRNAs in Living Cells.

In this work, the orderly aggregated catalytic hairpin assembly (OA-CHA) was developed for synchronous ultrasensitive detection and high-efficiency colocalization imaging of dual-miRNAs by a carefully designed tetrahedral conjugated ladder DNA structure (TCLDS). Exactly, two diverse hairpin probes were fixed on tetrahedron conjugated DNA nanowires to form the TCLDS without fluorescence response, which triggered OA-CHA in the aid of output DNA 1 and output DNA 2 produced by targets miRNA-217 and miRNA-196a cycle to generate TCLDS with remarkable fluorescence response. Impressively, compared with the traditional CHA strategy, OA-CHA avoided the fluorescence group and quenching group from approaching again because of the spatial confinement effect to significantly enhance the fluorescence signal, resulting in the simultaneous ultrasensitive detection of dual-miRNAs with detection limits of 21 and 32 fM for miRNA-217 and miRNA-196a, respectively. Meanwhile, the TCLDS with lower diffusivity could achieve accurate localization imaging for reflecting the spatial distribution of dual-miRNAs in living cells. The strategy based on OA-CHA provided a flexible and programmable nucleic amplification method for the synchronous ultrasensitive detection and precise imaging of multiple biomarkers and had potential in disease diagnostics..

SDR-assisted multi-primers triggering functional signal hairpin probe for miRNA detection with enzyme-free and high sensitivity

MiRNA can exist in body fluids and its changes directly affect human life and health, which has been a research hotspot in recent years. In this work, we designed an enzyme-free sensing system for highly sensitive detection of miRNA-21 based on strand displacement reaction (SDR) that can recycle the target and functional hairpin probes (HP) that modified on upconversion nanomaterials (UCNPs). The target gene circulates through the SDR process and completes the initial signal amplification process. Then, the C2 sequences and targets released by the SDR process can continue to be combined with functional hairpin probes modified with fluorophore and quenching groups. After infrared irradiation, HP is broken, and C2 or the target can take the fluorophores away from the quenching group, so that the fluorescence signal can be detected to achieve highly sensitive detection of the target. In sensing system, the use of magnetic beads can reduce the background signal during the reaction process, and the functional hairpin probe can accurately turn the signal on and off. The sensor system not only shows excellent detection performance (detection limit of 30.3 fM) in buffer and actual detection, but also has excellent selectivity, which can provide a new detection method for miRNA-21 detection.

Ultrasensitive detection of nucleic acid based on a novel isothermal amplification

The present study describes a novel technology referred to as reciprocal two single-stranded DNA (ssDNA) probe-mediated isothermal amplification (TPIA) that enables target nucleic acid detection. TPIA extends the ssDNA probe A into ultralong ssDNA products with tandem repeats of hairpin and ssDNA probe B to form double-stranded DNA hairpins of varying lengths via continuously repeated polymerization and displacement. The entire TPIA process was supervised in real time with the assistance of fluorescence techniques. By exploiting this new mechanism, the system can be used as an autocephalous module to provide a versatile tool for a biosensing program. The ssDNA probe B was relationally designed to form a stem-loop structure (HP), which can only unwind through specific recognition by the target nucleic acid. The HP stem region was then exposed and HP was rearranged to serve as the ssDNA probe B in the TPIA system after HP unfolding. The novel design strategy was exploited to specifically assay different target microRNAs (miR-21 and miR-221) with a low limit of detection on the 10 and 1 fM level, respectively. This method has great potential for nucleic acid detection in biomedical research and clinical diagnosis by rationally redesigning the template.

Target aided self-assembly of DNA hyperbranched nanostructures for bacterial 16 S ribosomal DNA gene SERS detection

Quantity analysis of bacterial 16 S rDNA gene is still a challenge. Here we successfully achieved bacterial 16 S rDNA gene SERS analysis with target aided self-assembly of DNA hyperbranched nanostructures. Catalytic hairpin assembly (CHA) was first introduced for self-assembly of branched nanostructures, including target bacterial DNA served as trigger probe and three hairpin probes for further target activated cross opening. Rolling cycle amplification (RCA) was then introduced by using a circular DNA as template and single stranded branches of the formed branched nanostructures as primer, forming a concatemer containing multiple copies of tandem repeating segments with same sequences complementary to the circular templates. After that, thousands of Raman-Au nanoprobe would hybridize this long single stranded DNAs for DNA-AuNPs nanoaggretates assembly. By using SERS signal collection, target bacterial DNAs would be successfully analyzed with ultra-sensitivity as low as 15.0 fM. Furthermore, selectivity test for targets with mismatched DNA sequences and other bacterial 16 S rDNA genes show good results. This sensing method was extended to quantify target genes in human plasma samples, showing its excellent applicability in complex samples.

DNAzyme-Mediated Biodeposition Coupling Adjustable Cascade Electric Fields for Photoelectrochemical Telomerase Activity Monitoring.

Telomerase, as a specialized reverse transcriptase, plays a vital role in early cancer diagnostics and prognosis; thus, developing efficient sensing technologies is of vital importance. Herein, an innovative "signal-on-off" photoelectrochemical (PEC) sensing platform was developed for ultrasensitive evaluation of telomerase activity based on an electron-transfer tunneling distance regulation strategy and DNAzyme-triggerable biocatalytic precipitation. Concretely, cascade internal electric fields between CuInS2 quantum dots (QDs), graphitic carbon nitride nanosheets (g-C3N4 NSs), and TiO2 nanorod arrays (NRAs) were developed to realize cascade electron extraction and hole transfer. Enabled by such a design, an effective "signal-on" state to gain a progressively enhanced PEC output was designed by suppressing the photogenerated electron-hole pair recombination. With the introduction of hairpin probe H2 and the subsequent extension of the primer sequence driven by the target telomerase, the CuInS2 QDs labeled with hairpin probe H1 were programmatically unfolded, resulting in CuInS2 QDs' close proximity to the working electrode away from the cascade interface, accompanied by the formation of G-quadruplex/hemin complexes. The gradual undermining of tunneling distance and implantation of DNAzyme-initiating biocatalytic precipitation tremendously induced the sluggish migration kinetics of the photoinduced charge, accompanied by the photocurrent intensity decrement, leading to the "signal-off" state. Under optimized conditions, the as-prepared PEC biosensor realizes ultrasensitive detection of telomerase activity from 10 to 105 cell·mL-1 with a detection limitation of 3 cells·mL-1. As a proof of concept, this well-designed method provides new insights into signal amplification for telomerase activity evaluation and also presents promising potential for further development in drug screening, healthcare diagnostics, and biological assays.

Navigable DNA walker coupled with exonucleases-assisted signal amplification for sensitive fluorescent detection of Staphylococcus aureus

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) was constructed by integrating the mechanisms of fluorescence signal regulation and exonucleases-assisted two-step signal amplification into a navigable DNA walker. After the recognition of S. aureus, the first signal amplification step converts the aptamer-cDNA1 duplex to the hydrolysate of cDNA1 by exonuclease I (Exo I). Then the second signal amplification step converts the hydrolysate to the fuel chain of the navigable DNA walker with the help of exonuclease III (Exo III) reaction system containing hairpin probe and cDNA2. Driven by the fuel chain, the movement of the DNA walker triggers the fluorescence resonance energy transfer (FRET) to obtain an amplified fluorescent signal that correlates to the target concentration. The movement track is ingeniously designed and constructed by DNA self-assembly technology, which controls the motion direction and stride of the DNA walker. In addition, the movement triggers FRET, which effectively enhances the detection signal. The biosensor has a wide detection concentration range of 1 × 102-2 × 107 CFU/mL, and the detection limit is 9 CFU/mL. This biosensor provides a reference method for customization of signal amplification strategy in DNA circuit machinery. And has potential applications in the fields of environmental and food safety monitoring.

Hairpin cascade circuits on programmable DNA-nucleated spherical nucleic acid for intracellular targeted microRNA imaging

DNA molecular probes have been increasingly used for biological imaging and tumor theranostics. In particular, live-cell imaging of low abundance microRNA (miRNA) with high accuracy and efficiency was extremely important for understanding functions and dynamics of miRNA. Herein, an easy-to-obtain DNA-nucleated spherical nucleic acid (DSNA) was constructed by one pair of hairpin probes “sticked” on self-assembled three-dimensional (3D) urchin-like DNA nanostructures (UDNs) to realize sensitive imaging of tumor-related miRNA in living cells. UDNs were prepared through four short single-stranded DNA by a simple “one-pot” annealing process that took only a few minutes. Moreover, sticking the reactants of hairpin probes on the UDNs not only sped up the reaction kinetic rate, but also improved the sensitivity and efficiency of miRNA-triggered hairpin cascade circuits. Compared with traditional AuNP-based spherical nucleic acid, the DSNA possessed superb biocompatibility and stability, excellent cell internalization capability and accessibility, which endowed them tremendous advantages for the monitoring of miRNA in living cells. Therefore, our developed strategy exhibited great potential in sensitive imaging of intracellular miRNA, offering a new avenue for early tumor diagnosis and related biomedical research.

A Simple Ratiometric Electrochemical Aptasensor Based on Exonuclease III-Assisted Target Recycling for Ultrasensitive Detection of Prostate Specific Antigen

Prostate specific antigen (PSA) is considered as an important biomarker in prostate cancer diagnosis. Herein, an ultrasensitive ratiometric electrochemical aptasensor was constructed for analyzing PSA. The ferrocene (Fc)-labeled thiolated hairpin probe 2 (Fc-HP2) was conjugated to the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface by Au-S bonds. With the introduction of PSA, the aptamer region of hairpin probe 1 (HP1) preferred to bind with PSA, and the rest of HP1 could hybridize with Fc-HP2 to form Fc-HP2/HP1/PSA complex with a blunt 3′ terminus, which triggered the exonuclease III (Exo III) cleavage process accompanied by Fc leaving from the electrode and recycling of HP1/PSA. The remaining Fc-HP2 fragments left on the electrode surface were then hybridized with methylene blue-labeled DNA (MB-DNA). This resulted in an enhancement of MB signal (IMB) and a decrement of Fc signal (IFc). Under the optimal conditions, the proposed aptasensor showed good analytical performance for sensitive detection of PSA with a linear range from 100 fg·mL−1 to 10 ng·mL−1 and a detection limit of 34.7 fg·mL−1. The fabricated aptasensor had been applied to detect PSA in diluted human serum samples and obtained satisfied results, suggesting it had great potential in clinical diagnosis.

Spherical nucleic acid enzyme programmed network to accelerate CRISPR assays for electrochemiluminescence biosensing applications

Given the targeted binding ability and cleavage activity of the emerging CRISPR/Cas12a assay which transduces the target into its cleavage activity exhibited broadly prospective applications in integrated sensing and actuating system. Here, we elaborated a universal approach to quickly activate CRISPR/Cas12a for low-abundance biomarker detection based on the amplification strategy of a target-induced spherical nucleic acid enzyme (SNAzyme) network that could accelerate the output of activators. Specifically, multifunctional Y-shaped probes and hairpin probes (HPs, which contained the specific sequence of the activators of CRISPR/Cas12a and the substrate chain of DNAzyme) were rationally designed to construct SNAzyme. Target recognition induced disassembly of the Y-shaped probes, which released DNAzyme strands to active DNAzyme and accompanied by SNAzyme self-assembly into SNAzyme network. Interestingly, compared with randomly dispersed SNAzyme, the reaction kinetics of the SNAzyme network enhanced 1.6 times in response to Α-methyl acyl-CoA racemase (AMACR, a biomarker for prostate cancer), which was attributed to the promoted catalytic efficiency of DNAzyme by the confined SNAzyme network. Benefiting from these, the prepared biosensor based on electrochemiluminescence (ECL) platform by loading AuAg nanoclusters (AuAgNCs) into metal-organic framework-5 (MOF-5) exhibited satisfying detection performance for AMACR with a wide linear range (0.001 μg/mL to 100 μg/mL) and a low detection limit (1.0 × 10−4 μg/mL, which exhibited significant potential in clinical diagnoses.

A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

Trigger-activated autonomous DNA machine for amplified liver cancer biomarker microRNA21 imaging.

MicroRNA-21 (miRNA-21) is a kind of RNA that exists in biological fluids such as blood, urine and saliva. It has over expression in liver cancer and has different expression in different stages of cancer. However, due to the characteristics of small base number, short length, low abundance and easy degradation of miRNA-21, the detection of miRNA-21 is a challenging subject. Visualization, sensitive, specific and stable detection of tumor suppressor or oncogene microRNAs (miRNAs) remains challenging and is highly significant for clinical diagnostics. To solve this problem, we have developed a target-triggered hybridization assembly DNA machine for intracellular miRNA imaging based on strand displacement amplification (SDA) and branched hybridization chain reaction (B-HCR). In this approach, the target miRNA could hybridize with the template probe to trigger the SDA, resulting in the formation of nicked fragments (NFs) that hybridized with hairpin probe1 (HP1). The opened HP1 could hybridize with hairpin probe2 (HP2), leading to the self-assembly of hyperbranched DNA nanostructures through B-HCR. As expected, the newly developed method exhibits a detection limit down to 11.3pM miRNA-21 and achieves high selectivity toward miRNA-21 against other interfering miRNAs. Due to its superior sensitivity and selectivity, our method can be further used to detect miRNA-21 in human serum samples. By taking advantage of intelligent design, the proposed method was also used for image miRNA-21 expression levels in different cell lines. This method shows a broad application in clinical diagnosis.

Enzyme-assisted amplification of target cycle triggers the unlocking of locked hairpin probes for let-7a detection

The detection of miRNA in cells is difficult owing to its substantially low cellular content. Therefore, developing a highly sensitive sensor to detect cellular miRNA remains a significant challenge. Herein, we report an enzyme-assisted biosensor with target cycle amplification that can trigger the unlocking of locked hairpin probes for sensitive and robust let-7a gene detection. In the research, three kinds of hairpin probes were skillfully designed. The hairpin probe comprises a complementary sequence of a target, primer, and recognition site of Nt. BbvCI restriction endonucleases. In addition, the alternating synergistic impact of polymerase and the nicking enzyme generates considerable triggers to unlock the locked hairpin probe LH1, consequently triggering a subsequent circulating strand displacement reaction to form a stable H1–H2 double strand to ensure sufficient distance between a fluorophore on H1 and a quenching group on bolt DNA (bDNA), and resulting in the recovery of fluorescence. Furthermore, this process does not require complicated operation procedures and instruments, and the target gene let-7a can be sensitively detected. Specifically, the detection limit of the biosensor is as low as 160 fM, and its linear range is 0.5 pM–250 nM. Moreover, this biosensor can be employed to detect let-7a in human serum with good selectivity.

Low-Cost Full-Range Detection of C-Reactive Protein in Clinical Samples by Aptamer Hairpin Probes and Coprecipitation of Silver Ions and Gold Nanoparticles.

C-reactive protein (CRP) levels can vary widely related to diverse disease contexts. However, expensive antibodies have impeded the clinical utility of antibody-based full-range CRP assays, especially in developing countries. Herein, we established a low-cost, antibody-free, 96-well plate-based full-range CRP detection method by combining gold nanoparticles (AuNPs), silver iodide (AgI), Eosin Y, and the aptamer hairpin probe (AHP) with Ag+-mediated cytosine-cytosine mismatches, that is, the Au@AgI/Eosin Y-AHP method. After binding the target CRP, the AHP released Ag+, which subsequently induced the aggregation of AuNPs on the surface of AgI colloids, resulting in a significant increase in the adsorption of Eosin Y on the surface of AuNPs. The changes in fluorescence intensity (FI) of Eosin Y in the supernate without and with CRP were proportional to the concentration of the CRP in the wide range of 0.01-40 ng/mL (r = 0.9969), and 96 samples can be detected in 96-well plates simultaneously by a microplate reader within 45 min. Remarkably, the CRP levels of 100 clinical samples achieved with the Au@AgI/Eosin Y-AHP had a good correlation with those obtained with the latex-enhanced immune turbidimetry assay (r = 0.986). Furthermore, the kit based on the Au@AgI/Eosin Y-AHP method costs only $8.1 for 100 tests. Therefore, the new method is beneficial for less developed areas where expensive assays are not affordable.

Construction of an Oxidative Cleavage‐Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity

AbstractMyeloperoxidase (MPO) is a mammalian pro‐oxidant protease and it is closely related to severe infections and diverse inflammatory diseases. Rapid and sensitive measurement of MPO activity is essential for anticancer drug discovery and inflammatory research. Herein, we demonstrate the construction of an oxidative cleavage‐activated deoxyribozymes (DNAzyme) biosensor for rapid detection and cellular imaging of the MPO activity. When target MPO is present, the phosphorothioate (PS)‐modified hairpin probe is site‐specifically cleaved by MPO, releasing the intact Mg2+‐dependent DNAzyme sequences. Subsequently, the activated DNAzyme initiates the cyclic cleavage of the signal probe with the assistance of cofactor Mg2+, liberating large numbers of Cy5 molecules. This assay possesses the characteristics of easy operation, low sample consumption, without the requirements of expensive radiolabeling, antibodies, and nanomaterials. Especially, this assay can be performed in one pot under isothermal conditions (37°C) within 60 min. Due to the high efficiency of DNAzyme‐based cyclic cleavage reaction and the intrinsic advantages of single‐molecule detection, this assay achieves high sensitivity with a limit of detection (LOD) of 2.74×10−3 ng μL−1. It can be applied to screen MPO inhibitors, measure cellular MPO activity at the single‐cell level, and image intracellular MPO in living cells, providing a powerful platform for early clinical diagnosis and drug discovery.